Methods and devices for collecting, handling and processing mammary fluid samples for evaluating breast diseases, including cancer

ABSTRACT

Biological samples of mammary fluid or components thereof are obtained using a breast pump device coupled with a solid phase sample collection medium, alternatively facilitated by administering oxytocin to the subject. The breast pump device stimulates expression of mammary fluid and provides for collection of diagnostic samples to evaluate breast disease, including cancer. The biological sample may include whole cells or cellular components, purified or bulk proteins, glycoproteins, peptides, nucleotides or other desired constituents comprising a breast disease marker. Methods, kits and adapter devices relating to the breast pump device are also provided. Yet additional methods, devices, accessories, and materials are provided for laboratory handling and processing of breast fluid samples and for related diagnostic methods.

RELATED APPLICATIONS

This application claims the priority benefits of U.S. Provisional PatentApplication No. 60/248,134, filed by Quay on Nov. 13, 2000 and U.S.Provisional Patent Application No. 60/248,136, filed by Quay on Nov. 13,2000. The disclosures of each of the foregoing priority applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD OF INVENTION

The invention relates to methods, devices, and kits for obtaining andassaying biological samples from mammary fluid. More specifically, theinvention relates to methods, devices, and kits for obtaining andassaying fluid and cytological samples from the mammary glands of amammalian subject for evaluating, diagnosing and managing breastdisease, including infections, pre-cancerous conditions, cancersusceptibility and cancer.

BACKGROUND OF THE INVENTION

Breast cancer is by far the most common form of cancer in women, and isthe second leading cause of cancer death in humans. Despite many recentadvances in diagnosing and treating breast cancer, the prevalence ofthis disease has been steadily rising at a rate of about 1% per yearsince 1940. Today, the likelihood that a women living in North Americawill develop breast cancer during her lifetime is one in eight.

The current widespread use of mammography has resulted in improveddetection of breast cancer. Nonetheless, the death rate due to breastcancer has remained unchanged at about 27 deaths per 100,000 women. Alltoo often, breast cancer is discovered at a stage that is too faradvanced, when therapeutic options and survival rates are severelylimited. Accordingly, more sensitive and reliable methods are needed todetect small (less than 2 cm diameter), early stage, in situ carcinomasof the breast. Such methods should significantly improve breast cancersurvival, as suggested by the successful employment of Papinicolousmears for early detection and treatment of cervical cancer.

In addition to the problem of early detection, there remain seriousproblems in distinguishing between malignant and benign breast disease,in staging known breast cancers, and in differentiating betweendifferent types of breast cancers (e.g. estrogen dependent versusnon-estrogen dependent tumors). Recent efforts to develop improvedmethods for breast cancer detection, staging and classification havefocused on a promising array of so-called cancer “markers.” Cancermarkers are typically proteins that are uniquely expressed (e.g. as acell surface or secreted protein) by cancerous cells, or are expressedat measurably increased or decreased levels by cancerous cells comparedto normal cells. Other cancer markers can include specific DNA or RNAsequences marking deleterious genetic changes or alterations in thepatterns or levels of gene expression associated with particular formsof cancer.

A large number and variety of breast cancer markers have been identifiedto date, and many of these have been shown to have important value fordetermining prognostic and/or treatment-related variables. Prognosticvariables are those variables that serve to predict disease outcome,such as the likelihood or timing of relapse or survival.Treatment-related variables predict the likelihood of success or failureof a given therapeutic plan. Certain breast cancer markers clearly serveboth functions. For example, estrogen receptor levels are predictive ofrelapse and survival for breast cancer patients, independent oftreatment, and are also predictive of responsiveness to endocrinetherapy. Pertschuk et al., Cancer 66:1663-1670, 1990; Parl and Posey,Hum. Pathol. 19:960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056,1989; Anderson and Poulson Cancer 65:1901-1908, 1989.

The utility of specific breast cancer markers for screening anddiagnosis, staging and classification, monitoring and/or therapypurposes depends on the nature and activity of the marker in question.For general reviews of breast cancer markers, see Porter-Jordan et al.,Hematol. Oncol. Clin. North Amer. 8:73-100, 1994; and Greiner,Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in thesereviews, a primary focus for developing breast cancer markers hascentered on the overlapping areas of tumorigenesis, tumor growth andcancer invasion. Tumorigenesis and tumor growth can be assessed using avariety of cell proliferation markers (for example Ki67, cyclin D1 andproliferating cell nuclear antigen (PCNA)), some of which may beimportant oncogenes as well. Tumor growth can also be evaluated using avariety of growth factor and hormone markers (for example estrogen,epidermal growth factor (EGF), erbB-2, transforming growth factor (TGF),which may be overexpressed, underexpressed or exhibit altered activityin cancer cells. By the same token, receptors of autocrine or exocrinegrowth factors and hormones (for example insulin growth factor (IGF)receptors, and EGF receptor) may also exhibit changes in expression oractivity associated with tumor growth. Lastly, tumor growth is supportedby angiogenesis involving the elaboration and growth of new bloodvessels and the concomitant expression of angiogenic factors that canserve as markers for tumorigenesis and tumor growth.

In addition to tumorigenic, proliferation and growth markers, a numberof markers have been identified that can serve as indicators ofinvasiveness and/or metastatic potential in a population of cancercells. These markers generally reflect altered interactions betweencancer cells and their surrounding microenvironment. For example, whencancer cells invade or metastasize, detectable changes may occur in theexpression or activity of cell adhesion or motility factors, examples ofwhich include the cancer markers Cathepsin D, plasminogen activators,collagenases and other factors. In addition, decreased expression oroverexpression of several putative tumor “suppressor” genes (for examplenm23, p53 and rb) has been directly associated with increased metastaticpotential or deregulation of growth predictive of poor disease outcome.

Additional representative breast disease markers within these variousclasses include prostaglandin E2 (PGE2); estrogen-regulated proteinssuch as pS2; interleukins (e.g., IL-10); S-100 protein; vimentin;epithelial membrane antigen; prostate specific antigen (PSA); bcl-2;CA15-3 (an aberrant form of polymorphic epithelial mucin (PEM)); CA19-9; mucin core carbohydrates (e.g., Tn antigen and Tn-like antigens);alpha-lactalbumin; lipidassociated sialic acid (LASA);galactose-N-acetylgalactosamine (Gal-GaINAC); GCDFP-15; Le(y)-relatedcarbohydrate antigen; CA 125; urokinase-type plasminogen activator (uPA)and uPA related antigens and complexes (e.g., LMW-uPA, HMW-uPA, uPAaminoterminal fragment (ATF), uPA receptor (UPAR) and complexes withinhibitors such as PA1-1 and PA1-2); beta-glucuronidase; CD31; CD44splice variants; blood group antigens (e.g., ABH, Lewis, and MN); andgenetic lesions or altered expression levels of CCND1, EMS1, BRCA1 andBRCA2 genes.

In summary, the evaluation of proliferation markers, oncogenes, growthfactors and growth factor receptors, angiogenic factors, proteases,adhesion factors and tumor suppressor genes, among other cancer markers,can provide important information concerning the risk, presence, statusor future behavior of cancer in a patient. Determining the presence orlevel of expression or activity of one or more of these cancer markerscan aid in the differential diagnosis of patients with uncertainclinical abnormalities, for example by distinguishing malignant frombenign abnormalities. Furthermore, in patients presenting withestablished malignancy, cancer markers can be useful to predict the riskof future relapse, or the likelihood of response in a particular patientto a selected therapeutic course. Even more specific information can beobtained by analyzing highly specific cancer markers, or combinations ofmarkers, which may predict responsiveness of a patent to specific drugsor treatment options.

Methods for detecting and measuring cancer markers have been recentlyrevolutionized by the development of immunological assays, particularlyby assays that utilize monoclonal antibody technology. Previously, manycancer markers could only be detected or measured using conventionalbiochemical assay methods, which generally require large test samplesand are therefore unsuitable in most clinical applications. In contrast,modern immunoassay techniques can detect and measure cancer markers inrelatively much smaller samples, particularly when monoclonal antibodiesthat specifically recognize a targeted marker protein are used.Accordingly, it is now routine to assay for the presence or absence,level, or activity of selected cancer markers by immunohistochemicallystaining breast tissue specimens obtained via conventional biopsymethods. Because of the highly sensitive nature of immunohistochemicalstaining, these methods have also been successfully employed to detectand measure cancer markers in smaller, needle biopsy specimens whichrequire less invasive sample gathering procedures compared toconventional biopsy specimens. In addition, other immunological methodshave been developed and are now well known in the art which allow fordetection and measurement of cancer markers in non-cellular samples suchas serum and other biological fluids from patients. The use of thesealternative sample sources substantially reduces the morbidity and costsof assays compared to procedures employing conventional biopsy samples,which allows for application of cancer marker assays in early screeningand low risk monitoring programs where invasive biopsy procedures arenot indicated.

For the purpose of breast cancer evaluation, the use of conventional orneedle biopsy samples for cancer marker assays is often undesirable,because a primary goal of such assays is to detect the cancer before itprogresses to a palpable or mammographically detectable tumor stage.Prior to this stage, biopsies are generally contraindicated, makingearly screening and low risk monitoring procedures employing suchsamples untenable. Therefore, there is general need in the art to obtainsamples for breast cancer marker assays by less invasive means thanbiopsy, for example by serum withdrawal.

Efforts to utilize serum samples for breast cancer marker assays havemet with limited success, largely because the targeted markers areeither not detectable in serum, or because telltale changes in thelevels or activity of the markers cannot be monitored in serum. Inaddition, the presence of breast cancer markers in serum probably occursat the time of micro-metastasis, making serum assays less useful fordetecting pre-metastatic disease. In contrast, fluid within the mammaryglands themselves is expected to contain much higher and morebiologically relevant levels of breast cancer markers than serum,particularly in view of the fact that 80%-90% of all breast cancersoccur within the intraductal epithelium of these glands. Fluid withinthe breast ducts is expected to contain an assemblage and concentrationof hormones, growth factors and other potential markers comparable tothose secreted by, or acting upon, the surrounding cells of thealveolar-ductal system. Likewise, mammary fluid is expected to containcells and solid cellular debris or products that can be used incytological or immunological assays to evaluate intracellular or cellsurface markers that may not be detectable in the liquid fraction ofmammary fluid.

Previous attempts to develop non-invasive breast cancer marker assaysutilizing mammary fluid samples have included studies of mammary fluidobtained from patients presenting with spontaneous nipple discharge. Inone of these studies, conducted by Inaji et al., Cancer 60:3008-3013,1987, levels of the breast cancer marker carcinoembryonic antigen (CEA)were measured using conventional, enzyme linked immunoassay (ELISA) andsandwich-type, monoclonal immunoassay methods. These methodssuccessfully and reproducibly demonstrated that CEA levels inspontaneously discharged mammary fluid provide a sensitive indicator ofnonpalpable breast cancer. In a subsequent study, also by Inaji et al.,Jpn. J. Clin. Oncol. 19:373-379, 1989, these results were expanded usinga more sensitive, dry chemistry, dot-immunobinding assay for CEAdetermination. This latter study reported that elevated CEA levelsoccurred in 43% of patients tested with palpable breast tumors, and in73% of patients tested with nonpalpable breast tumors. CEA levels in thedischarged mammary fluid were highly correlated with intratumoral CEAlevels, indicating that the level of CEA expression by breast cancercells is closely reflected in the mammary fluid CEA content. Based onthese results, the authors concluded that immunoassays for CEA inspontaneously discharged mammary fluid are useful for screeningnonpalpable breast cancer.

Although the evaluation of mammary fluid has been shown to be a usefulmethod for screening nonpalpable breast cancer in women who experiencespontaneous nipple discharge, the rarity of this condition renders themethods of Inaji et al, inapplicable to the majority of women who arecandidates for early breast cancer screening. In addition, the firstInaji report cited above determined that certain patients sufferingspontaneous nipple discharge secrete less than 10 μl of mammary fluid,which is a critically low level for the ELISA and sandwich immunoassaysemployed in that study. It is likely that other antibodies used to assayother cancer markers may exhibit even lower sensitivity than theanti-CEA antibodies used by Inaji and coworkers, and may therefore notbe adaptable or sensitive enough to be employed even in dry chemicalimmunoassays of small sampbs of spontaneously discharged mammary fluid.

In view of the above, an important need exists in the art for morewidely applicable, non-invasive methods and materials to obtainbiological samples for use in evaluating, diagnosing and managing breastdisease including cancer, particularly for screening early stage,nonpalpable breast tumors. A related need exists for methods andmaterials that utilize such readily obtained biological samples toevaluate, diagnose and manage breast disease, particularly by detectingor measuring selected breast cancer markers, or panels of breast cancermarkers, to provide highly specific, cancer prognostic and/ortreatment-related information, and to diagnose and manage pre-cancerousconditions, cancer susceptibility, breast infections and other breastdiseases.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to providenon-invasive methods and kits for obtaining biological samples that canbe employed in assays for evaluating, diagnosing and managing breastdisease, particularly cancer.

It is a further object of the invention to achieve the above object inassay methods and kits that are widely applicable to a broad range ofpatients, and that include useful assays and kits for screening earlystage, nonpalpable mammary tumors.

It is yet another object of the invention to provide methods and kitsthat utilize the aforementioned biological samples to evaluate, diagnoseand manage breast disease, preferably breast cancer, by detecting and/ormeasuring selected breast disease markers such as breast cancer markers,or panels of breast cancer markers, to provide highly specificprognostic and/or treatment-related information to the clinician.

The invention achieves these objects and other objects and advantagesthat will become apparent from the description which follows byproviding non-invasive methods and devices for obtaining biologicalsamples from a mammary organ of a mammalian patient. Through the use ofnovel, specialized breast pump devices of the invention, which arefluidly connected with (i.e., by direct or indirect coupling) a solidphase sample collection medium, the physician can rapidly andnon-invasively collect mammary fluid samples from lactating ornon-lactating female patients without additional intervention. Alternatemethods of the invention for mammary fluid sample collection may involveadministration of oxytocin, or an oxytocin analog, in an amounteffective to stimulate or increase expression of mammary fluid inducedin conjunction with employment of the breast pump device. The oxytocinor oxytocin analog (for example a long-acting oxytocin analog such ascarbetocin) is administered in a manner (e.g., intranasally) and amountsufficient to reach and stimulate a target alveolar-ductal tissue of thebreast, whereby the oxytocin stimulates myoepithelial contraction of thealveolar-ductal tissue to induce or facilitate mammary fluid expression.Alternatively, an intramuscular or intravascular injection of oxytocincan effect the same myoepithelial contraction response as the intranasaladministration route. The amount, timing and/or mode of oxytocinadministration may be adjusted on an individual basis depending on suchfactors as menstrual cycle stage, use of birth control or hormonereplacement therapy, pregnancy history, age of onset of menarche,ethnicity and other factors known to affect an individual's propensityfor breast fluid expression.

Mammary fluid collection devices of the invention are effective toinduce mammary fluid expression for sample collection, alone or inconjunction with oxytocin stimulation. These devices are typicallyprovided as a specialized breast pump which can be applied to the breastcovering the nipple, and which typically directly receives the expressedmammary fluid. In preferred methods involving use of a breast pump,negative pressure is generated on the breast to induce expression ofmammary fluid, optionally facilitated by prior or concurrentadministration of oxytocin. In yet additional alternative methods,mammary fluid can be expressed and collected without the aid of a breastpump, which may require an increase of oxytocin dosage or lengthening ofthe post administration time period before the mammary fluid is fullyexpressed from the nipple.

During or after mammary fluid expression, a biological sample iscollected from the expressed mammary fluid, which sample may consist ofwhole mammary fluid, whole cells, cell fragments, cell membranes,selected liquid, cellular or other solid fractions of the mammary fluid,as well as proteins, glycoproteins, peptides, nucleotides (including DNAand RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid.

Sample collection can be achieved simply by receiving the expressedmammary fluid within any suitable reservoir, such as an ordinary samplestorage container or assay vessel. In preferred embodiments of theinvention, the expressed mammary fluid is exposed to a solid phasesample collection medium, simultaneous with or subsequent to the time ofbreast fluid expression. Suitable solid phase media in this contextinclude microscopic glass slides, capillary tubes, coated tubes,microtiter wells or plates, membranes, filters, affinity columns, dotblot matrices, beads, microspheres, resins, and other like media thatwill selectively adsorb, bind, filter, partition or otherwise processdesired components of the mammary fluid for convenient incorporationinto a desired assay. Often it will be desirable to combine a pluralityof solid phase media for sample collection, e.g., a filter and membrane,a membrane and a particulate medium, etc., for example to differentiallypartition and adsorb selected components of the breast fluid.

In conjunction with sample collection, the sample may be exposed toother agents such as buffers, diluents, extraction or chromatographicmedia, cross-linking agents, denaturing agents, etc., to stabilize orotherwise prepare the sample for processing within a desired assay.

Thus provided within the invention are methods and devices for obtaininga biological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid, whichemploy a novel breast pump or breast pump adapter. The breast pumpfunctions in a similar fashion as a conventional breast pump but alsoprovides a solid phase sample collection medium in fluid connection withthe pump. The solid phase sample collection medium may be integratedwithin the breast pump or otherwise fluidly connected therewith, so thata sample of expressed mammary fluid contacts the collection medium,typically while the pump remains applied to the breast. In more detailedaspects of the invention, methods for employing the novel breast pumpinclude a step of applying the breast pump to stimulate breast fluidexpression, with or without prior oxytocin or carbetocin induction,wherein the solid phase sample collection medium is fluidly connectedwith a breast engaging portion or member of the breast pump.

According to these methods, operation of the pump results in anexpressed breast fluid sample contacting the solid phase samplecollection medium, typically while the pump remains applied to thebreast. Within the foregoing methods, additional methods are providedwhich employ a novel, hand-held breast pump device, wherein a doctor,technician or patient collecting a breast fluid specimen can grasp andoperate the hand-held pump to stimulate expression of breast fluid andcollect a specimen thereof while keeping one hand free for additionaltasks. The compact hand-held pump design allows the device to be pickedup and manipulated with one hand, to seat the breast engaging elementagainst the breast, apply vacuum pressure to the breast by manualoperation of the vacuum pump to cause a suitable volume of breast fluidto be expressed at or near the nipple, and to simultaneously collect atleast a primary sample of expressed breast fluid onto, or within, thesolid phase sample collection medium without additional manual steps ora need to remove the device from the breast or engage two hands in theoperation.

In certain collection methods of the invention, breast fluid expressedby use of the general purpose or hand-held breast pump is simultaneouslyor subsequently diluted, filtered, washed, admixed with fixative orother processing agents, or otherwise processed or modified to yield acollected fluid sample partially or completely devoid of cells, proteinsand/or other selected components originally present in the expressedfluid, to provide a processed fluid sample for laboratory analysis. Inother embodiments, particulate components of the breast fluid, forexample, cells, cellular components and/or cellular debris, arecollected after processing and/or modification, e.g., for cytologicalexamination. Often, primary sample collection and/or processing in thiscontext is coincident with the fluid contacting one or more solid phasecollection medium(a) fluidly connected with the breast engaging member.Depending on the type(s) of medium(a) used, preliminary sampleprocessing can also be achieved directly by simple operation of thehand-held pump, without the need for additional processing steps orremoval of the breast engaging member from the subject's breast.

In other alternative methods within the invention, preliminary sampleprocessing involves additional steps following breast fluid expression.In certain embodiments, the breast engaging member is removed from thebreast after the breast fluid is expressed and the fluid is transferredto a first solid phase sample collection medium, typically a membrane orfilter. This initial or primary stage of sample collection may befollowed by washing or by manual transfer of selected breast fluidcomponents (e.g., proteins, carbohydrates, cells, or cellular debris)from the first solid phase collection medium (e.g., a nitrocellulosemembrane) to a second solid phase medium, e.g., a fluid-containingreservoir. Typically, preliminary sample processing in this regardprecedes final packaging of the collected sample for storage or shipmentto a lab for further processing and analysis of the sample. In oneexample, whole cells or other cellular materials are separated fromexpressed mammary fluid onto a nitrocellulose membrane or a filter,which is typically secured in fluid connection with the breast engagingmember by a fixed or removable support member mounted to the engagingmember or sample collection housing. The cells are subsequentlytransferred or washed in fluid (e.g., cytology fluid) to a second solidphase sample collection medium, for example a removable fluid reservoirconnected to, or integrated with, the breast engaging member or samplecollection housing.

In relation with these methods, various sample collection devices andaccessories for use therewith are provided within more detailedembodiments of the invention. Typically, breast fluid collection devicesof the invention include a breast engaging member constructed of anon-porous material that is sized and dimensioned to receive at least anipple portion of a human breast and form a suction seal therewith. Oneor more solid phase sample collection media are provided in fluidconnection with the breast engaging member for receiving a sample ofexpressed breast fluid. A vacuum pump mechanism is provided in gaseousconnection with the breast engaging member for generating negativepressure through the breast engaging member to facilitate breast fluidexpression.

In specific embodiments of the collection device of the invention, asample collection housing is fluidly connected with the breast engagingmember. The solid phase sample collection medium is often removablysupported within the housing in proximity to the nipple when the breastengaging member is applied to the breast and negative pressure isgenerated by the vacuum pump mechanism. The solid phase samplecollection medium can be one or more microscopic glass slides, capillarytubes, collection tubes, vials, columns, micro-columns, wells, plates,membranes, filters, resins, inorganic matrices, beads, resins,particulate chromatographic media, plastic microparticles, latexparticles, coated tubes, coated templates, coated beads, or coatedmatrices.

Optional features of the breast fluid collection device includeremovable coupling means for removably coupling said sample collectionhousing with said breast engaging member. In other embodiments, thesolid phase sample collection medium may be supported by a supportmember integrally or removably mounted within the sample collectionhousing in fluid connection with said breast engaging member. Varioustypes of support members, including disposable or reuseable discs,cartridges and cassettes are provided as an accessory for use within theinvention. In yet additional embodiments, a reciprocating mechanism forreciprocally adjusting a position of the solid phase sample collectionmedium relative to the breast engaging member is incorporated within thedevice. The reciprocating mechanism may incorporate a support member orcarrier reciprocatingly mounted relative to the breast engaging member,which support member or carrier supports the solid phase samplecollection medium. Yet another optional feature of the device includes abreast pump adapter employing concepts of the invention for collectionof mammary fluid samples and operable in combination with a conventionalbreast pump.

In other detailed embodiments of the invention, the sample collectiondevice is a hand-held breast pump incorporating the breast engagingmember and the vacuum pump mechanism in a compact, structurallyintegrated breast fluid collection apparatus suitable for manipulationand operation using only one hand. In certain embodiments, handheldbreast pump comprises a modular device made up of a plurality ofcomponents, each joined or securable in fixed structural interconnectionwith one another and capable of partial or complete disassembly fromremaining components to facilitate operation, cleaning, servicing and/orstorage of the device. The modular breast pump can include, for example,a separate breast engaging member detachable from one or moreinterconnecting components of the device for cleaning or to allowinterchanging of different engaging members to accommodate breastanatomy differences among patients.

Within more detailed embodiments of the hand-held breast pump, the solidphase sample collection medium can be supported by a support memberremovably mounted in fluid connection with the breast engaging member.The support member can be a removable cassette for removable placementin fluid connection with the breast engaging member. The support membercan house any of the above identified collection media, and mayincorporate one or more air channels that pass through a body of thesupport member for passage of vacuum pressure and/or sample materialsbetween the breast engaging member and a sample collection housingmember of the hand-held breast pump.

Within other detailed embodiments, the hand-held device includes afluid-retaining recess, well or reservoir integrated or fluidlyconnected with the support member or a sample collection housing memberof the device. The fluid-retaining recess, well or reservoir maycomprise an integral, defined compartment or enclosure within the samplecollection housing for receiving the breast fluid and/or constituentsamples thereof. Alternatively, fluid-retaining recess, well orreservoir comprises a removable fluid reservoir member of the samplecollection housing, typically provided as a rigid sample collection tubeor vial removably connected with an outer casing member of the housing.The removable reservoir member is optionally sealably connected with theouter casing member of the housing to form an airtight couplingtherewith. In certain embodiments, the removable reservoir memberfeatures a circumferential ridge, fin, O-ring or other sealableengagement means to engage and make an airtight seal against a wall orother surface of the casing member when the vial is nested within thecasing member.

In additional detailed embodiments, the removable reservoir member isgaseously and fluidly connected with the breast engaging member tofacilitate sample collection. For example, the vacuum pressure from thevacuum pump means may be routed to the breast engaging member throughthe removable reservoir member of the housing, which is modified toinclude one or more air ports that form a gaseous connection between alumen of the reservoir and the vacuum pump means. The reservoir membermay function in this context as both a conduit for vacuum pressuretransmission to the breast and a receptacle for fluid sample materialsto directly collect expressed fluid or as a secondary collection mediumto receive primarily collected sample materials washed or otherwisetransferred from a primary solid phase sample collection medium into thereservoir. For example, a primary solid phase sample collection mediumfluidly connected with the breast engaging member may be positioned tocollect a primary sample of one or more breast fluid components whichcan thereafter be washed or otherwise transferred directly or indirectlyinto the removable reservoir member, without removal or disassembly ofthe breast engaging member and reservoir member.

The fluid collection reservoir may serve a dual purpose for collection,as well as for storage, transport and/or processing of collected breastfluid or breast fluid component samples. Relating to this purpose, theremovable reservoir member further comprises closure means for closingthe reservoir after sample collection is completed to prevent samplecontamination and spillage. The closure means may comprises a capadapted to sealably engage a top end of the removable reservoir member.Where the reservoir member is modified to include one or more air portsfor transmission of vacuum pressure between the lumen of the reservoirand the vacuum pump means, the closure means include secondary closuremeans to sealably close the air port(s) after sample collection. Forexample, the secondary closure means may comprise an adhesive seal orsticker sized and constructed to adhere to an outer wall of thereservoir member surrounding an air port opening. Typically, thesecondary closure means comprises a combined closure and labeling devicewhich functions as a secondary closure mechanism to seal the air port(s)of the removable reservoir, and as a labeling template to provide awriting surface for sample labeling. The combined closure and labelingtab or sticker generally includes a first, closure-forming surface forapplication over the air port to form a seal by juxtaposition oradhesive contact with an outer wall of the removable reservoir, and asecond, labeling surface opposite the closure-forming surface made of ablank template material suitable for receiving a stable, ink or graphiteimprint. In more detailed aspects, the secondary closure means comprisesa combined closure and labeling tab or sticker which is pre-attached tothe removable reservoir member in a first, open configuration and whichcan be manually repositioned or otherwise manipulated after samplecollection to a second, closed configuration to form a seal or closureagainst the air port(s).

In related aspects of the invention, a novel breast fluid collectionreservoir, e.g., a modified cytology vial, is provided for use within amammary fluid collection device of the invention, which reservoirincorporates the foregoing features of the removable reservoir member ofthe sample collection housing. The novel collection reservoir thusprovided is useful within the breast fluid collection methods of theinvention, as well as within sample processing and diagnostic assaymethods performed in the laboratory subsequent to collection of a breastfluid sample.

In related aspects of the invention, methods are provided fordetermining the presence or amount of a breast disease marker,preferably a breast cancer marker, in biological samples obtained from amammary organ of a mammalian patient. These methods may involveintranasal, intramuscular or intravascular administration of oxytocin oran oxytocin analog to mammalian patients in amounts effective tostimulate mammary fluid expression in the patient. Once a sufficientpost-administration time period has elapsed to allow the oxytocin toreach and stimulate target alveolar-ductal tissues, mammary fluid iscollected directly from the nipple or, alternatively, the breast ispumped, and a biological sample from expressed mammary fluid iscollected, as above. After the sample is collected a bioassay isconducted on the sample to determine the presence and/or amount of thebreast disease marker in the sample. Suitable bioassays in this regardinclude assays to detect known markers of breast disease, such as assaysemploying immunological or other suitable probes to detect specificantigens and other markers expressed by selected pathogens, includingbacterial and viral pathogens. More preferred bioassays will detectindividual markers or panels of markers of benign breast tumors,pre-cancerous breast disease, and/or breast cancer, such as assaysemploying immunological or other suitable probes to detect specificantigens and other markers expressed by benign, pre-cancerous and/orcancerous alveolar-ductal cells of the breast. Preferably, the assaywill detect the presence or amount of multiple breast disease markers inthe biological sample, for example by including a panel of immunologicalor molecular probe(s) that bind or react with multiple breast cancermarkers.

Within further related aspects of the invention, novel methods areprovided handling or processing a biological sample of mammary fluid, ora component thereof, for use in a diagnostic assay to detect or quantifya breast disease marker in a biological sample of mammary fluid. Themethods typically include providing or obtaining the biological sampleof mammary fluid, or component thereof, in a fluid-retaining reservoirthat features a top end defining a primary opening for access to thesample and an outer reservoir wall defining one or more air ports thatcommunicate between the outer wall and an inner lumen of the vial. Incertain aspects, the fluid-containing reservoir member includes closuremeans for closure of the reservoir after the sample is introducedtherein, to prevent sample contamination and spillage.

The closure means may comprise a cap adapted to sealably engage a topend of the removable reservoir member. In various embodiments, theclosure includes secondary closure means to sealably close the airport(s) after the biological sample is introduced into said reservoir.Exemplary secondary closure means include an adhesive seal or stickerthat is sized and constructed to adhere to an outer wall of thereservoir member surrounding an air port opening. Alternatively, thesecondary closure means may include a combined closure and labelingdevice which functions as a secondary closure mechanism to seal the airport(s) of the removable reservoir and as a labeling template to providea writing surface for sample labeling. The combined closure and labelingtab or sticker may be directly applied to seal the air port after samplecollection having a first, closure-forming surface for application overthe air port to form a seal by juxtaposition or adhesive contact with anouter wall of the removable reservoir, and a second, labeling surfaceopposite the closure-forming surface made of a blank template materialsuitable for receiving a stable, ink or graphite imprint thereon.

Within additional aspects of the invention, the first, closure-formingsurface of the reservoir can be provided with an adhesive coatingresistant to disruption by contact with aqueous solutions. The secondaryclosure means may include a combined closure and labeling tab or stickerwhich is pre-attached to the removable reservoir member in a first, openconfiguration and which can be manually repositioned or otherwisemanipulated after sample collection to a second, closed configuration toform a seal or closure against the air port(s). The secondary closuremeans can optionally comprise an adhesive tab or strip folded in theopen configuration to form an inner layer affixed to the reservoirproximate to the air port, and an outer layer folded over the innerlayer, said outer layer providing the first, closure-forming surface andthe second, labeling surface, wherein the outer layer can be unfoldedaway from the inner layer and wrapped around the reservoir so that theclosure-forming surface covers the air port to form a fluid-resistantclosure and the labeling surface faces outward for recordation of sampledata. The outer layer can be optionally secured in a folded-backposition against the inner layer by adhesive engagement of the labelingsurface with the inner layer.

In related embodiments, the first, closure-forming surface may beprovided with an adhesive coating that is protected in the openconfiguration by folding of an end segment of the outer layer bearingthe adhesive coating back, so that the closure forming surface providesa protective surface to shield the adhesive prior to closure, wherebythe end segment can be lifted and pulled outward to unfold the endsegment to separate the adhesive coating on the closure-forming surfacefrom the protective surface and to release the outer layer from theinner layer for closing of the air port(s).

In certain aspects of the invention, the foregoing methods utilize afluid-retaining reservoir that is a modified cytology vial. In otheraspects, the fluid-retaining reservoir comprises a removable fluidreservoir member of a sample collection housing of a mammary fluidcollection device. The fluid-retaining reservoir may be a rigid samplecollection tube or vial that is optionally removably connected with anouter casing member of a sample collection housing of said collectiondevice. The fluid-retaining reservoir can alternately be adapted forremovable, sealable connection with the outer casing member of thehousing to form an airtight coupling therewith. In exemplaryembodiments, the fluid-retaining reservoir is a cytology vial that issealably connectable with said outer casing member to form an airtightcoupling therewith. The fluid-retaining reservoir can optionally beremovably nested within the casing member to form a substantiallyairtight contact between an inner wall of the casing member and an outerwall, or a top or bottom end, of the reservoir member. In certainconfigurations, an outer wall of the fluid-retaining reservoir bears acircumferential ridge, fin or O-ring. The fluid-retaining reservoir canoptionally comprise a removable fluid reservoir member of the samplecollection housing of a mammary fluid collection device, and theabove-described circumferential ridge, fin or O-ring may be adapted toengage and make a circumferential airtight seal against an inner wall ofa casing member of the sample collection housing of the device.

In exemplary sample handling and processing methods of the invention,the mammary fluid sample or component thereof includes whole cells orcell fragments. The handling and processing methods can further includethe step of accessing the sample within reservoir to transfer or processthe sample for detection or quantification of the breast disease marker.In exemplary embodiments, the sample may be transferred to a secondreservoir or other sample container or template for processing thesample to detect or quantify the breast disease marker. Exemplary stepsof sample processing include detection or quantification of the breastdisease marker by staining cells or cell fragments from the sample witha cytological stain to detect a cytological marker. Alternativeprocessing steps include detection or quantification of the breastdisease marker by microscopic examination of stained cells or cellfragments from the sample. Within certain embodiments, the biologicalsample includes one or more components selected from the groupconsisting of whole mammary fluid, whole cells, cell fragments, cellmembranes, purified proteins, bulk proteins, glycoproteins, peptides andpolynucleotide components of a primary mammary fluid sample. Often, thebreast disease marker will comprise a breast cancer marker. In variousaspects, the breast disease marker is selected from the group consistingof a protein, a peptide, a glycoprotein, a lipid, a DNA polynucleotideand an RNA polynucleotide. Exemplary breast disease markers include Ki67Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen,Transforming Growth Factor, Tissue Plasminogen Activator, Insulin GrowthFactor Receptors, Collagenase Type IV, Laminin Receptor, Integrins, p53,rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-SpecificEnolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HMFG, MCA,vasopressin and cathepsin.

Within the foregoing methods the breast disease markers may be detectedusing any of a variety of useful techniques, for example by ELISAimmunoassay, immunoprecipitation assay, or solid phase immunoassay.

In yet additional aspects of the invention, clinically useful kits areprovided for determining the presence and/or amount of a breast diseasemarker, preferably a breast cancer marker, in biological samplesobtained from a mammary organ of a mammalian patient. The kits include amammary fluid collection device in the form of a general purpose orhand-held breast pump as described herein. Additional kits include oneor more breast pump attachments (e.g., a detachable breast engagingmember, or multiple such attachments for use with different patients),accessories (e.g., replaceable fluid-retaining reservoirs), solid phasemedia, and/or disposable or reusable support members, cartridges orcassettes for holding collection media, as described herein. These andother kit components may be provided, alone or in any combination, withor without inclusion of the basic breast pump apparatus in the kit. Yetadditional kits include a pharmaceutical preparation of oxytocin or anoxytocin analog in a biologically suitable carrier for use in alternatemammary fluid collection methods of the invention. Still other kitsinclude on or more preparative and/or diagnostic reagents selected fromthose disclosed herein, including one or more fixatives, probes, labelsand the like in separate or common containers. In certain embodiments ofthe invention, kits include compositions and/or devices for detectingthe presence or amount of one or more breast disease marker(s) in thebiological sample, often including one or more immunological ormolecular probe(s) that binds or reacts with one or more breast cancermarker(s). The foregoing kit components are generally assembled in acollective packaging unit, which may include written or otherwiseuser-accessible instructions detailing the sample collection, handlingand/or processing methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial sectional view of a breast pump employing theconcepts of the invention.

FIG. 2 is a sectional view of a portion of the breast pump as indicatedin FIG. 1.

FIG. 3 is a perspective view of a support member for supporting a solidphase sample collection medium in fluid connection with a breast pump.

FIG. 4 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 5 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 6 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 7 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 8 is a partial sectional view of a breast pump device employing theconcepts of the invention.

FIG. 9 is a partial sectional view of a portion of a breast pumpillustrating a support member and cartridge for containing a particulatesolid phase sample collection medium.

FIG. 10 is a partial sectional view of a portion of a breast pumpillustrating a support member and an exemplary solid phase samplecollection template (coated tube).

FIG. 11 is a partial sectional view of a breast pump employing areciprocating mechanism to adjust positioning of a solid phase samplecollection medium within the pump.

FIG. 12 is a sectional view depicting a breast pump adapter employingthe concepts of the invention.

FIG. 13 is a sectional view depicting a breast pump adapter employing areciprocating mechanism to adjust positioning of a solid phase samplecollection medium within the adapter.

FIGS. 14 and 15 provide partial sectional views of a breast pumpemploying a sliding reciprocating mechanism to adjust positioning of asolid phase sample collection medium within the pump.

FIG. 16 is a sectional view illustrating a hand-held breast pump of theinvention.

FIG. 17 is a sectional view illustrating a hand-held breast pump of theinvention.

FIG. 18 is an exploded perspective view illustrating a hand-held breastpump of the invention employing a removable support member and removablefluid reservoir for primary and secondary sample collection.

FIGS. 19 and 20 provide perspective views of alternative embodiments ofa removable sample collection reservoir for use with the hand-heldbreast pump of the invention.

FIGS. 21-23 provide sectional views of the removable fluid reservoir ofFIG. 19, illustrating operation of a closure/labeling strip to seal thereservoir and provide an exterior labeling surface for recordation ofsample data.

FIGS. 24-26 provide top plan views of a modified membrane or filter foruse within the breast pump of the invention having perforations or slitsfor enhancing permeability and flexibility of the membrane or filter.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As noted above, the invention provides methods and devices 10, 10′ forobtaining, handling, and processing biological samples from mammaryfluid. Preferably, these methods are non-invasive, meaning they arenon-surgical and do not involve penetration of the breast by needles orother intrusive devices. To practice the non-invasive sample collectingmethod, the invention provides specialized breast pump devices whichfeature a breast engaging portion or member coupled with a vacuum pumpmechanism and fluidly connected with a solid phase sample collectionmedium. The mammary fluid collection devices and methods disclosedherein are related in certain aspects to mammary fluid collectiondevices and methods disclosed in U.S. patent application Ser. No.09/435,131, filed Nov. 5, 1999, U.S. patent application Ser. No.09/027,362, filed Feb. 20, 1998, and U.S. Pat. No. 5,798,266, issuedAug. 25, 1998, each incorporated herein by reference. The devices andmethods of the present invention are effective to induce mammary fluidexpression for sample collection, alone or in conjunction with oxytocinstimulation.

The mammary fluid collection devices of the invention are typicallyprovided as a specialized breast pump 10, 10′ that can be applied to ahuman or animal breast covering the nipple, and which typically receivesexpressed mammary fluid within a solid phase sample collection medium,and in some cases within a removable sample collection reservoir. Duringor after mammary fluid expression, a biological sample is collected fromthe expressed mammary fluid, which sample may consist of whole mammaryfluid, whole cells, cell fragments, cell membranes, selected liquid,cellular or other solid fractions of the mammary fluid, as well asproteins, glycoproteins, peptides, nucleotides (including DNA and RNApolynucleotides) and other like biochemical and molecular constituentsof the mammary fluid.

The breast pump devices 10, 10′ of the invention function in part bygenerating negative pressure applied to the nipple area of the breast toinduce mammary fluid expression. Fluid expression induced by thesebreast pump devices may optionally be facilitated by coordinateadministration of the peptide hormone oxytocin, or a functional analogthereof, in an amount effective to stimulate or increase expression ofmammary fluid induced by the breast pump device. The oxytocin oroxytocin analog (for example a long-acting oxytocin analog such ascarbetocin) is administered in a manner (e.g., intranasally) and amountsufficient to reach and stimulate a target alveolar-ductal tissue of thebreast, whereby the oxytocin stimulates myoepithelial contraction of thealveolar-ductal tissue to induce or facilitate mammary fluid expression.

During or after the mammary fluid expression step, a biological sampleis collected from the expressed mammary fluid. A range of suitablebiological samples are contemplated and will be useful within themethods of the invention, including whole mammary fluid, selected liquidor solid fractions of the mammary fluid, whole cells or cellularconstituents, proteins, glycoproteins, peptides, nucleotides (includingDNA and RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid. Sample collection can be achievedsimply by receiving the expressed mammary fluid within a suitablereservoir, such as an ordinary sample storage container or assay vessel.

In preferred embodiments of the invention, the expressed mammary fluidis contacted with a solid phase sample collection medium fluidlyconnected with the breast pump 10, 10′, simultaneous with or subsequentto the time of breast fluid expression. Suitable solid phase media inthis context include microscopic glass slides, capillary tubes, coatedtubes, microtiter wells or plates, membranes, filters, affinity columns,dot blot matrices, beads, resins, and other like media that willselectively adsorb, bind, filter, partition or otherwise process desiredcomponents of the mammary fluid for convenient incorporation into adesired assay.

A wide range of sample collection procedures and materials known in theart are useful within the invention. Selected methods and materials willvary among different assays, as will be understood and readily practicedby those skilled in the art. For example, if the breast disease markersought in a particular assay is a soluble protein, it will often bedesired to immobilize the protein on a solid phase matrix or template bycontacting the target protein with a reagent having high specificity forthe protein, preferably a polyclonal or monoclonal antibody. The yieldsa complex, e.g., a ligand-protein complex, an antibody-antigen complex,or other complex in which the target protein is bound to a specificbinding partner (i.e., wherein the complex is not dissociated uponaddition of a non-specific binding partner conventionally used as acontrol to determine specific binding; and preferably wherein thebinding partner binds with an affinity of kD 10-9 or greater). Thebinding partner that binds to the target protein is in turn immobilizedto the solid phase medium, before or after complex formation with thetarget protein. Immobilization of the binding partner, e.g., by covalentbinding to a solid phase template or matrix, can be achieved by avariety of conventional methods known in the art.

In this manner, the target protein/binding partner complex is adsorbedor otherwise bound directly to an insoluble matrix. Alternatively, avariety of secondary binding partners, e.g., anti-isotype antibodies,may be added to bind the complex to the insoluble matrix. The latterstep depends on the nature of the first binding partner (i.e., thebinding agent that specifically binds the target protein), for examplewhether the first binding partner is a primary antibody, ligand, etc.

Particularly useful within the invention are immunoassay which formatsemploy a combination of solid phase or immobilized reagents and labeledreagents whereby the association of the label with the solid phase is afunction of the presence or absence of reactivity with the targetedantigen. In general, such a solid phase reagent comprises a bindingsubstance such as an anti-antibody (e.g., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, bound or attached, covalently or noncovalently, to the solidphase matrix or in an otherwise immobilized form.

Useful labeled reagents in solid phase immunoassays include a bindingsubstance such as an anti-antibody (e.g., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, which is chemically coupled with a detectable chemical moiety.Useful labels are conventional in the art and include fluorescers,chemiluminescers, radioisotopes, and enzymes. Enzyme labels areparticularly useful and are generally selected from alkalinephosphatase, peroxidase, and P-galactosidase. Enzyme labels are readilydetectable by addition of a corresponding chromogenic substrate anddetecting the resulting color or fluorescent response.

A variation of this protocol uses a ligand-modified form of the targetedantigen(s) with immobilization to the solid phase being accomplished byusing a solid phase bearing an immobilized (e.g., bound or adsorbed)binding partner to the ligand. For example, biotin or a hapten (e.g.,fluorescein) can be used as the ligand and can be immobilized by contactwith a solid phase form of avidin or anti-hapten antibody, respectively.The addition of the solid phase binding partner can occur at anyconvenient time in the assay, such as prior to contact of sample withthe ligand-antigens(s) or thereafter.

Preferred solid phase matrices for use within the foregoing methodsinclude Staphylococcus aureus or Protein A or G Agarose [e.g. Sepharose®(Pharmacia Biotech AB, Uppsala, Sweden)] beads. Protein A and protein Gare cell wall proteins isolated from specific bacterial strains, andhave specific binding sites for certain classes of immunoglobulins.Protein A binds (to varying degrees) most subclasses of IgG, plus IgM,IgA, and IgD. Protein G binds nearly all subclasses of IgG, but notother classes of immunoglobulins.

An alternative solid phase sample collection and/or assay methodutilizes a specific anti-marker primary antibody that is covalentlyattached to the solid phasematrix, e.g., by covalent linking theantibody through its free amino groups to cyanogen-bromide activatedSepharose particles. Insolubilized antibody can be used to pull thecorresponding marker antigen out of solution by adsorption to itssurface. In yet another alternative format, the marker protein can betreated with a cross-linking reagent (e.g. biotin or digoxigenin) thatmay be subsequently detected by a second binding partner. In the case ofbiotin, the second binding partner is avidin or streptavidin; fordigoxigenin, the second reagent is an anti digoxigenin antibody. Avidinand streptavidin may be coupled directly to the solid phase medium,e.g., to agarose beads. Because the initial biotinylation is notspecific for the marker, samples are frequently electrophoresed on,e.g., SDS PAGE, transferred to nitrocellulose etc., and Western blottedwith antibodies specific for the protein factor.

A preferred assay method for detecting protein markers is the wellknown, Enzyme Linked Immunosorbant Assay (ELISA) assay. According tothis method, a variety of coating reagents can be adsorbed or otherwisebound directly onto a surface of a desired solid phase sample collectionmedium, e.g., a microtiter plate, well, tube, bead, test strip, plasticmicroparticle, latex particle, etc., to form a coated template ormatrix. These coating reagents are typically a species-specificanti-isotype antibody (e.g., anti-mouse-IgG) but can also include ananti-marker primary antibody or an affinity reagent such as avidin orstreptavidin. The target protein (e.g., a soluble protein marker) iscontacted with a specific primary antibody or, alternatively, iscrosslinked (e.g., to biotin) or otherwise modified to form a complex,and the resulting complex is adsorbed to the coated template or matrixand processed according to conventional assay methods.

Latex or particle agglutination methods are also to be mentioned.Particles are coated or covalently coupled with a target antigen,ligand, antibody or other binding partner. The particles are thenincubated with a test sample and resulting agglutination of theparticles, e.g., due to formation of ICA antibody linkages betweenparticles, is detected. Detection can be accomplished by visualobservation (e.g., a slide agglutination format) or quantified bymeasuring turbidity changes with a spectrophotometer or nephelometer. Awell known variation of this general method based on inhibition ofparticle agglutination can also be employed. In addition, anagglutinator reagent can be prepared comprising multiple antigens, e.g.,a water soluble polymer backbone to which are attached multiples of oneor more antigens within a panel.

Alternative methods for collecting and analyzing samples within theinvention include Western immunoblot and dot-blot methods. Forapplication of these methods, the solid phase sample collection mediumis preferably a membrane or filter, e.g., a nitrocellulose,polyvinylidene difluoride (PVDF), or nylon membrane. Proteins within thebreast fluid sample may be processed (e.g., separated on SDS PAGE) ordirectly transferred to the membrane, and non-specific interactions maybe blocked by incubating the membrane with, e.g., bovine serumalbumin/ovalbumin or non-fat dry milk. A primary antibody withspecificity for the protein marker is contacted with the membrane, andexcess antibody is washed, e.g., with buffered detergent. A labeledisotype specific antibody is next contacted with the membrane, andtarget protein-primary antibody-secondary antibody ternary complexes aredetected, e.g., calorimetrically.

Where the targeted protein factor includes a carbohydrate moiety, thefactor can also be adsorbed to a solid phase template or matrix, e.g, aresin, by way of lectin-carbohydrate interactions. Various lectins areavailable for this purpose that differ in their carbohydrate bindingspecificity. For example, Lectin Con A binds to mannose-containingcarbohydrate structures and with low affinity to α-glucose andα-N-acetylglucosamine. Lectin GNA binds to terminal mannose residues.Lectin MAA binds to α(2-3) Linked sialic acids. A variety of otherlectins collectively providing a wide range of specificities are knownin the art.

A particularly preferred solid phase sample collection medium for usewithin the invention is a filter, pad or membrane that can be directlycontacted to a sample of expressed breast fluid to adsorb, absorb, bind,partition or otherwise facilitate sample processing or handling within aselected assay. For this purpose, several types of transfer membranesare known, including nitrocellulose that is the most commonly usedtransfer membrane. Several commercial sources now offer nitrocelluloseimpregnated with a synthetic support that improves its durability andflexibility without altering its performance. One preferred transfermembrane, polyvinylidene difluoride (PVDF), marketed by Millipore(Bedford, Mass.) under the trade name Immobilon®, has slightly lowerprotein-binding capacity than nitrocellulose but is mechanicallystronger and compatible with many organic solvents. This allows directprotein staining with Coomassie Blue, and direct amino acid compositionand sequence analysis of transferred proteins, without interfering withits subsequent use for antibody probing.

Membranes are not only useful within the invention for protein blotting,but also for immobilization of nucleic acids. Thus, nitrocellulose,reinforced nitrocellulose, diazotized membranes (paper or nylon), nylon,charged nylon, or PVDF, and DEAE-anion exchange membranes are useful forimmobilizing DNA and RNA from expressed breast fluid. In this context,the most commonly used membranes are reinforced nitrocellulose andnylon. Nitrocellulose has a lower background but also a lower bindingcapacity than nylon and is chosen primarily when background, but notdetectability, is the main concern. Nylon, in contrast, is ideal forlower copy number sequences, short target sequences (down to oligomers)or for reprobing. Membranes are also available with different poresizes. For DNA blots, membranes with a pore size of 0.45 μm are usuallychosen for large fragments, but 0.22 μm for fragments of <500 bases. ForRNA blots, membranes with a pore size of 0.1 or 0.22 μm are mostefficient. Membranes are available in different size specifications,including sheets, rolls, pre-cut circles, etc.

Methods for detecting DNA on nylon without DNA purification andprocessing of the samples, e.g., for detecting DNA from fluids or wholecells, have recently been developed (Reed and Matthaei, Nucleic AcidsRes. 18:3093, 1990; and Hammermueller et al., J. Virol. Methods 31:47,1991; each incorporated herein by reference). These procedures avoidenzymatic dispersion of cells, RNase and pronase treatments to hydrolyzecellular macro-molecules, etc., and are typically based on the capacityof alkali and other reagents to disperse and solubilize cells andhydrolyze macro-molecules including RNA and protein, but not DNA.Positively charged modified nylon membranes then irreversibly bindnucleic acid while remaining suitable for hybridization.

Nucleic acid extraction and processing steps may also be minimized bywell known fast blot methods. In particular, fast blot methods that usenylon as a solid phase take advantage of the ability of NaOH todissociate cells, denature DNA and immobilize DNA. Nitrocellulosemembranes have a lower binding capacity and co-immobilization of nucleicacid and protein from neutral solutions can be a problem. ConcentratedNaI can be used to inhibit protein immobilization, to denature DNA andto irreversibly bind the nucleic acid to nitrocellulose without arequirement for baking. This method can also be used for RNA.

Although it is possible to directly transfer proteins, nucleic acids andother markers to a solid phase matrix which is in turn directlyincorporated in an assay, it may be desirable to concentrate the targetmarker, e.g., by chromatography, extraction, specific or nonspecificadsorption, etc., particularly when sensitivity is a problem. Thus,samples can be collected and initially processed by contacting breastfluid with a solid phase chromatographic medium, e.g., within acartridge comprising a micro-column of Sepharose-coupled antibody. Up to500-fold increases in immunoassay sensitivity with apparent recoveriesof 85 to 95% can be achieved using this approach. This and other wellknown chromatographic procedures provide a powerful approach to thequantitation of substances too dilute to be measured by routine methods.

For sample collection and processing using chromatographic and relatedmethods, a particulate solid phase sample collection medium ispreferred. Various particulate media are known which selectively adsorb,absorb, bind, or partition components of biological samples, which mediaare readily adapted for collection and processing of breast fluidsamples. These particulate can be coupled with various coating reagentsknown in the art, e.g., affinity reagents, to provided a coated medium,or may be used in an unmodified form.

Exemplary particulate sample collection media for use within theinvention include beads, plastic microparticles, latex microspheres,glass materials such as controlled porous glass, granular agarose basedmaterials, cross-linked dextran polymers, inorganic or organic ionexchanger materials, kieselsur and other silicate materials. Suitablematerials additionally include cellulosic materials, e.g.,diethylaminoethyl (DEAE) cellulose or diethylamino (DEA) cellulose. Alsouseful are natural polymeric carbohydrates and their syntheticallymodified, cross-linked or substituted derivatives, such as agar, agaroseand cross-linked dextran polymers.

Synthetic polymers which can be prepared with suitably porousstructures, such as vinyl polymers (e.g., polyethylene, polypropylene,polystyrene, polyvinylchloride, polyvinylacetate and its partiallyhydrolysed derivatives, polyacrylates, polyacrylamides,polymethacrylates), copolymers and terpolymers of the above vinylmonomers among themselves and with other monomers, polycondensates(e.g., polyesters and polyamides), and addition polymers, such aspolyurethanes or polyepoxides are also useful.

Yet additional particulate media are prepared from inorganic materialshaving a suitably porous form, such as sulfates or carbonates ofalkaline earth metals and magnesium. Examples include barium sulfate,calcium sulfate, calcium carbonate, magnesium carbonate, silicates ofalkali and alkaline earth metals and/or aluminum and/or magnesium, andaluminum or silicon oxides or hydrates, such as clays, alumina, talc,kaolin, zeolite, among others.

Also included among useful solid phase sample collection media porousbarrier materials suitable for use with breast pump and breast pumpadapter devices of the invention, for example to enclose particulatesolid phase media within a cartridge adapted for coupling in fluidconnection with a breast pump or breast pump adapter. Such porousbarrier materials are inert to and nonreactive with markers and otheranalytes and reagents used in assaying for breast disease markers, andare porous with respect to the passage of liquids and/or particulates ofa pre-selected size. Suitable materials include various porous materialssuch as nylon fabric, polyethylene and other plastic films, membranes,filters, glass wool, sponge, styrofoam, ceramic and other porousmaterials.

In conjunction with sample collection, samples of expressed breast fluidmay be exposed to other agents such as buffers, diluents, extraction orchromatographic media, cross-linking agents, blocking agents, denaturingagents, etc., to stabilize or otherwise prepare the sample forprocessing within a desired assay. For example, the sample may bediluted (e.g., by collecting the sample in a well or recess containingthe solid phase medium wetted or suspended in a diluent) to minimizenonspecific binding effects, e.g, affecting a subsequent immunoassay. Inthe exemplary context of sample collection for immunoassays, the avidityof the antibody for the marker antigen is an important consideration,whereby providing more or less diluent during sample collection andincubation may optimize a particular antigen-antibody system beingstudied.

Commonly used buffers for dilution include phosphate, borate, orTris-buffered saline. Usually, the choice of the buffer is notimportant. Nonetheless, a careful examination of the effect of buffer,pH, ionic strength, and divalent cations will facilitate use of a newsample collection/assay system in order to maximize sensitivity andresolve possible sources of interference within the assay. Althoughimmunoassays are usually carried out at neutrality, doing so is notalways optimal.

Nonspecific binding or adsorption, e.g., of antigens and haptens(especially hydrophobic haptens) to glass and plastic tubes or pipettesmay markedly influence measured activity in a particular immunoassay.With some proteins and polypeptides, nonspecific binding in immunoassaysis reduced if plastic tubes are used. The addition of protein to themedium may also minimize nonspecific adsorption and help avoiddenaturation of highly diluted antigens and antibodies. Therefore,assays involving iodinated antigens are generally carried out inprotein-containing buffers. Bovine serum albumin, gelatin, lysozyme, andovalbumin are commonly used, usually at final concentrations of 1 to 5mg/ml. In some systems diluted whole serum or proteins present in thesample itself are just as satisfactory. However, even though addedproteins are often beneficial, they should not be used indiscriminatelywithout making an evaluation for possible adverse effects, for examplecontaminating enzymes that may degrade the marker protein.

Other possible additives for improved sample collection and assaymethods, apart from buffer and protein, include enzyme inhibitors andchelating agents. In assays lasting longer than 3 days, a bacteriostaticagent, such as sodium azide, 0.1 to 0.2%, may also be incorporated intothe sample collection and/or assay medium to help avoid microbialgrowth.

Although a fundamental utility of the present invention lies in thenovel, noninvasive methods for obtaining biological samples from mammaryfluid, additional methods are disclosed herein that provide usefulassays for detecting and/or measuring important breast disease markersin these samples. In this context, the invention provides a broad rangeof assay methods incorporating known procedures and reagents fordetermining the presence and/or expression levels of breast diseasemarkers, particularly breast cancer markers, in biological samples. Asincorporated within the invention, these methods involve application ofa breast pump 10, 10′ to mammalian patients, optionally coupled withoxytocin administration in amounts effective to facilitate mammary fluidexpression in the patient. After the sample is collected, a bioassay isconducted on the sample to determine the presence and/or amount of aselected breast disease marker, preferably a breast cancer marker orpanel of breast cancer markers, in the sample.

As used herein, the term breast disease marker refers to any cell, cellfragment, protein, peptide, glycoprotein, lipid, glycolipid,proteolipid, or other molecular or biological material that is uniquelyexpressed (e.g. as a cell surface or secreted protein) by diseasedbreast cells, or is expressed at a statistically significant, measurablyincreased or decreased level by diseased breast cells, or in associationwith breast disease (e.g. a protein expressed by an infectious agentassociated with breast disease), or is expressed at a statisticallysignificant, measurably increased or decreased level by diseased breastcells compared to normal breast cells, or which is expressed bynon-diseased breast cells in association with breast disease (e.g. inresponse to the presence of diseased breast cells or substances producedtherefrom). Breast disease markers can also include specific DNA or RNAsequences marking a deleterious genetic change, or an alteration inpatterns or levels of gene expression significantly associated withbreast disease. Preferred breast disease markers include markers ofbreast infections, benign neoplasia, malignant neoplasia, pre-cancerousconditions, and conditions associated with an increased risk of cancer.

As used herein, the term breast cancer marker refers to a subset ofbreast disease markers, namely any protein, peptide, glycoprotein,lipid, glycolipid, proteolipid, or other molecular or biologicalmaterial that is uniquely expressed (e.g. as a cell surface or secretedprotein) by cancerous cells, or is expressed at a statisticallysignificant, measurably increased or decreased level by cancerous cellscompared to normal cells, or which is expressed by non-cancerous cellsin association with cancer (e.g. in response to the presence ofcancerous cells or substances produced therefrom). Breast cancer markerscan also include specific DNA or RNA sequences marking a deleteriousgenetic change, or an alteration in patterns or levels of geneexpression significantly associated with cancer. In addition, breastcancer markers can include cytological features of whole cells presentin mammary fluid, such as nuclear inclusions or cytoplasmic structuresor staining attributes uniquely expressed by, or associated with,cancerous cells.

Among the breast cancer markers that are useful within the methods ofthe invention, a subset are described in representative review articlesby Porter-Jordanet al., Hematol. Oncol. Clin. North Amer. 8:73-100,1994; and Greiner, Pharmaceutical Tech, May, 1993, pp. 28-44, eachincorporated herein by reference in its entirety. Other suitable markersare also widely known and can be readily incorporated into the methodsof the invention using information and methods generally known oravailable in the literature. Preferred breast cancer markers for usewithin the invention include well characterized markers that have beenshown to have important value for determining prognostic and/ortreatment-related variables in human female patients. As notedpreviously, prognostic variables are those variables that serve topredict outcome of disease, such as the likelihood or timing of relapseor survival. Treatment-related variables predict the likelihood ofsuccess or failure of a given therapeutic program. Determining thepresence or level of expression or activity of one or more of thesemarkers can aid in the differential diagnosis of patients with malignantand benign abnormalities, and can be useful for predicting the risk offuture relapse or the likelihood of response to a selected therapeuticoption.

It is important to note, however, that the invention does not relysolely on breast disease markers that meet the stringent requirements ofsensitivity and specificity that would render the marker immediatelyacceptable for clinical application to human patients. On the contrary,a number of breast disease markers contemplated within the inventionfall short of these stringent criteria, and nonetheless provide usefulinformation that can be of substantial benefit in detecting,differentially diagnosing or managing breast cancer. Such non-clinicallyaccepted markers are useful for immediate application within the methodsof the invention as basic research tools, and as adjunctive tools inclinical applications. Beyond these immediate applications, many suchmarkers are expected to be further developed and refined according tothe methods of the invention to the point of direct clinicalapplicability, particularly in assay methods that analyze combinationsof markers to generate complementary data of greater predictive valuethan data yielded by individual markers alone.

The preferred assay methods of the invention particularly focus onbreast cancer markers associated with tumorigenesis, tumor growth,neovascularization and cancer invasion, and which by virtue of thisassociation provide important information concerning the risk, presence,status or future behavior of cancer in a patient. As noted previously,tumorigenesis and tumor growth can be assessed using a variety of cellprolferation markers (for example Ki67, cyclin D1 and PCNA). Tumorgrowth can also be evaluated using a variety of growth factor andhormone markers (for example estrogen, EGF, erbB-2, and TGF-α, receptorsof autocrine or exocrine growth factors and hormones (for example IGFand EGF receptors), or angiogenic factors. In addition to tumorigenic,proliferation and growth markers, a number of markers provideinformation concerning cancer invasion or metastatic potential in cancercells, for example by indicating changes in the expression or activityof cell adhesion or motility factors. Exemplary markers in this contextinclude Cathepsin D, plasminogen activators and collagenases. Inaddition, expression levels of several putative tumor “suppressor”genes, including nm23, p53 and rb, provide important data concerningmetastatic potential, or growth regulation of cancer cells. A largenumber and variety of suitable breast cancer markers in each of theseclasses have been identified, and many of these have been shown to haveimportant value for determining prognostic and/or treatment-relatedvariables relating to breast cancer.

Prior to or concurrent with each assay run of the invention, it may bepreferable to perform a preliminary evaluation to verify sample originand/or quality. The focus of such preliminary evaluations is to verifythat the sample collected from expressed mammary fluid is indeed ofmammary origin, and is not contaminated with other potentialcontaminants, such as sweat from skin surrounding the nipple. For thesesample verification purposes, a variety of assays are available whichidentify mammary fluid markers known to be present in mammalian mammaryfluid, and which are preferably highly specific markers for mammaryfluid (i.e. markers which are typically always present in mammary fluidand which are absent from all, or most of, other potentiallycontaminating bodily fluids and tissues). However, an acceptable levelof specificity for mammary fluid markers within the methods of theinvention is provided by markers that are simply known to be present inmammary fluid, even though they may be present in other bodily fluids.One such marker is the enzyme lysozyme, which is a normal component ofhuman serum, urine, saliva, tears, nasal secretions, vaginal secretions,seminal fluid, and mammary fluid. Lysozyme (muramidase) is an enzymethat hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharidecell wall of a variety of microorganisms resulting in cell lysis.Quantitative measurement of lysozyme is readily accomplished by a wellknown agar plate diffusion method, described in detail in theinstructions provided with the Quantiplate® lysozyme test kit, availablefrom Kallestad, Sanofi Diagnostics (Chasta, Minn.), incorporated hereinby reference in its entirety.

Other mammary fluid markers for sample verification that are morespecific than lysozyme are preferred within the methods of theinvention, and can be readily incorporated within the invention based onpublished and generally known information. The most preferred amongthese markers are proteins and other biological substances that arespecifically expressed or enriched in mammary fluid. A diverse array ofsuitable markers in this context have been characterized and havealready been used to develop specific antibodies, including affinitypurified and monoclonal antibodies. These antibodies can in turn beemployed as immunological probes to determine the presence or absence,and/or to quantify, selected mammary fluid markers to verify mammaryfluid sample origin and quality. Mammary fluid markers of particularinterest for use within the invention include specific cytokeratins thatare characteristically expressed by normal and cancerous mammaryepithelial cells, against which specific panels of antibody probes havealready been developed. (See for example, Nagle, J., Histochem.Cytochem. 34:869-881, 1986, incorporated herein by reference in itsentirety). Also useful as mammary fluid markers are the human mammaryepithelial antigens (HME-Ags) corresponding to glycoprotein componentsof the human milk fat globulin (HMFG) protein, against which specificantibodies (e.g., anti HMFG1, Unipath, U.K.) are also available. (SeeRosner et al, Cancer Invest. 13:573-582, 1995; Ceriani et al. Proc.Natl. Acad. Sci. USA 74:582-586, 1982; Ceriani et al., Breast CancerRes. Treat. 15:161-174, 1990, each incorporated herein by reference inits entirety).

To conduct the breast disease marker assays provided within theinvention, a collected biological sample from mammary fluid is generallyexposed to a probe that specifically binds to a selected breast diseaseor breast cancer marker, or otherwise interacts with the marker in adetectable manner to indicate the presence or absence, or amount, of thebreast disease or breast cancer marker in the sample. Selected probesfor this purpose will generally depend on the characteristics of thebreast disease marker, i.e. on whether the marker is a proteinpolynucleotide or other substance. In preferred embodiments of theinvention, the breast disease marker is a protein, peptide orglycoprotein, all of which are effectively targeted in breast diseasemarker assays using specific immunological probes. These immunologicalprobes can be labeled with a covalently bound label to provide a signalfor detecting the probe, or can be indirectly labeled, for example by alabeled secondary antibody that binds the immunological probe to providea detectable signal.

General methods for the production of non-human antisera or monoclonalantibodies (e.g., murine, lagormorpha, porcine, equine) are well knownand may be accomplished by, for example, immunizing an animal with aselected breast disease marker protein, peptides synthesized to includepart of the marker protein sequence, degradation products including partof the marker protein sequence, or fusion proteins including all or partof the marker protein linked to a heterologous protein or peptide.Within various embodiments, monoclonal antibody producing cells areobtained from immunized animals, immortalized and screened, or screenedfirst for the production of an antibody that binds to the selectedbreast cancer marker protein or peptide, and then immortalized. It maybe desirable to transfer the antigen binding regions (i.e., F(ab′)2 orhypervariable regions) of non-human antibodies into the framework of ahuman antibody by recombinant DNA techniques to produce a substantiallyhuman molecule. Methods for producing such “humanized” molecules aregenerally well known and described in, for example, U.S. Pat. No.4,816,397 (incorporated herein by reference in its entirety).Alternatively, a human monoclonal antibody or portions thereof may beidentified by first screening a human B-cell cDNA library for DNAmolecules that encode antibodies that specifically bind to the selectedbreast disease marker according to the method generally set forth byHuse et al. (Science 246:1275-1281, 1989 (incorporated herein byreference in its entirety). The DNA molecule may then be cloned andamplified to obtain sequences that encode the antibody (or bindingdomain) of the desired specificity.

Also contemplated within the invention are bifunctional antibodieshaving independent antigen binding sites on each immunoglobulin molecule(as disclosed for example in Thromb. Res. Suppl. X: 83, 1990, and in TheSecond Annual IBC International Conference on Antibody Engineering, A.George ed., Dec. 16-18, 1991; each incorporated herein by reference inits entirety), as well as panels of individual antibodies havingdiffering specificities. Bifunctional antibodies and antibody panels ofparticular use within the invention include antibodies and panels ofantibodies that bind to two or more selected breast disease markers togenerate complementary data of greater predictive value than datayielded by individual markers alone.

Monoclonal antibodies are particularly useful within the invention aslabeled probes to detect, image and/or quantify the presence or activityof selected breast disease markers. In this context, monoclonalantibodies that specifically bind to selected breast disease markers areprovided which incorporate one or more well known labels, such as a dye,fluorescent tag or radiolabel. By incorporating such a label, theantibodies can be employed in routine assays to determine expression,localization and/or activity of one or more selected breast diseasemarkers in a biological sample including, or derived from, mammaryfluid. Results of these assays to determine expression, localizationand/or activity of a selected breast disease marker in a test sampletaken from a patient at risk for breast disease, or known to have breastdisease, can be compared to results from control studies detectingand/or quantifying the same marker in biological samples obtained fromnormal patients negative for breast disease. In this manner, baselinedata and cutoff values can be determined according to routine methods torefine the assays of the invention and adapt them for direct clinicalapplication.

Detection and/or quantification of breast disease markers in thebiological samples of the invention can be accomplished using a varietyof methods. Preferred methods in this regard include well known ELISAimmunoassays, immunoprecipitation assays, and various solid phaseimmunoassays including Western blotting, dot blotting and affinitypurification immunoassays, among other methods. Comparable methods aredisclosed herein, or are elsewhere disclosed and known in the art, forusing non-antibody probes to detect and/or quantify the expressionand/or activity of breast disease markers. Suitable non-antibody probesfor use within the invention include, for example, labeled nucleotideprobes that hybridize at moderate or high stringency to DNA transcriptsof oncogenes and other DNA sequences associated with elevated breastdisease risk, or with mRNA transcripts encoding breast disease markerproteins. Preferably, the nucleotide probes hybridize with a targetsequence under high stringency conditions. As used herein, “moderatestringency” and high stringency” refers to finite ranges ofhybridization conditions that are well established in the literature.(See, for example: Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, N.Y., Cold Spring Harbor Press, 1989; Hamesand Higgins, eds., Nucleic Acid Hybridization: A Practical Approach, IRLPress, Washington D.C., 1985; Berger and Kimmel, eds, Methods inEnzymology, Vol. 52, Guide to Molecular Cloning Techniques, AcademicPress Inc., New York, N.Y., 1987; and Bothwell, Yancopoulos and Alt,eds, Methods for Cloning and Analysis of Eukaryotic Genes, Jones andBartlett Publishers, Boston, Mass. 1990; each of which is incorporatedherein by reference in its entirety. Moderate or high stringencyhybridization conditions are achieved, e.g., by adjusting thetemperature of hybridization, adjusting the percentage ofhelix-destabilizing agents such as formamide in the hybridization mix,and adjusting the temperature and salt concentration of the washsolutions. Alternatively, stringency can be adjusted duringpost-hybridization washes by varying the salt concentration and/or thetemperature. Stringency of hybridization may be reduced by reducing thepercentage of formamide in the hybridization solution or by decreasingthe temperature of the wash solution. Typical high stringency conditionsrequire, for example, high temperature hybridization (e.g., 65-68° C. inaqueous solution containing 4-6× SSC, or 42° C. in 50% formamide)combined with a high temperature (e.g., 5-25° C. below the Tm) wash anda low salt concentration (e.g., 0.1× SSC). In contrast, moderatestringency conditions involve, for example, hybridization at atemperature between 50° C. and 55° C. and washes in 0.1× SSC, 0.1% SDSat between 50° C. and 55° C., which should be sufficient to identifypolynucleotide molecules encoding I-mf from other species or to isolateisoforms of I-mf. In further contrast, low stringency conditionsinvolve, for example, low hybridization temperatures (e.g., 35-42° C. in20-50% formamide) and intermediate temperature (e.g., 406° C.) washes ina higher salt concentration (e.g., 2-6× SSC).

In certain preferred embodiments of the invention, cDNA andoligonucleotide probes are employed in well known Northern, Southern anddot-blot assays for identifying and quantifying the level of expressionof a selected breast disease marker in cell samples collected fromexpressed mammary fluid.

Other suitable probes for use within the invention include labeledligands, binding partners and co-factors of breast disease markers (e.g.growth factor receptor ligands, or substrates of breast cancerassociated proteases such as cathepsin D).

Measuring the level of expression of breast disease markers according tothe foregoing methods will provide important prognostic andtreatment-related information for assessing a broad range of breastdisease, including the genesis, growth and invasiveness of cancer, inmammals, particularly humans. For example, assays utilizingoligonucleotide probes will assist early screening to evaluate heritablegenetic lesions associated with breast cancer, and to distinguishbetween precancerous, early cancerous and likely metastatic lesions inpatients.

In addition to the above mentioned sample collection and assay methods,the invention also provides kits and multicontainer units comprisingdevices, components, accessories, reagents and other related materialsfor practicing the sample collection and assay methods of the invention.These kits are clinically useful for collecting, handling and/orprocessing mammary fluid samples, e.g., for determining the presenceand/or amount of a breast disease marker, preferably a breast cancermarker, in the biological samples. The kits include a mammary fluidcollection device having a breast engaging member, vacuum pump, andsample collection means incorporated in a general purpose or hand-heldbreast pump as described herein. Additional kits include one or morebreast pump attachments (e.g., a detachable breast engaging member, ormultiple such attachments for use with different patients), accessories(e.g., replaceable fluid-retaining reservoirs), solid phase media,and/or disposable or reusable support members, cartridges or cassettesfor holding collection media, as described herein. These and other kitcomponents may be provided, alone or in any combination, with or withoutinclusion of the basic breast pump apparatus in the kit. Yet additionalkits include a pharmaceutical preparation of oxytocin or an oxytocinanalog in a biologically suitable carrier for use in alternate mammaryfluid collection methods of the invention. Preferably, the oxytocinpreparation is provided in an intranasal spray applicator and containsapproximately 40 USP units of oxytocin per ml of liquid carrier, whichcarrier is a simple, inexpensive buffered saline solution. Preferredapplicators can be in any of a variety of pressurized aerosol orhand-pump reservoir forms, with a nozzle for directing a liquid spray ofthe oxytocin into a patient's nostril. Still other kits include on ormore preparative and/or diagnostic reagents selected from thosedisclosed herein, including one or more fixatives, probes, labels andthe like in separate or common containers. In certain embodiments of theinvention, kits include compositions and/or devices for detecting thepresence or amount of one or more breast disease marker(s) in thebiological sample, often including one or more immunological ormolecular probe(s) that binds or reacts with one or more breast cancermarker(s). The foregoing kit components are generally assembled in acollective packaging unit, which may include written or otherwiseuser-accessible instructions detailing the sample collection, handlingand/or processing methods of the invention.

Kits for practicing the assay methods of the invention include asuitable container or other device for collecting, storing, handlingand/or processing a biological sample from expressed mammary fluid. Arange of suitable collection devices is contemplated corresponding to awide range of suitable biological samples that may be collected from theexpressed mammary fluid. For example, simple sterile containers orreservoirs are provided to collect whole mammary fluid. Alternatively, avariety of solid phase devices, including microscopic glass slides,membranes, filters, beads and like media, are provided to receive orpartition selected liquid or solid fractions of the mammary fluid, toreceive or partition cells or cellular constituents from the mammaryfluid, or to receive or partition purified or bulk proteins,glycoproteins, peptides, nucleotides (including DNA and RNApolynucleotides) or other like biochemical and molecular constituentsfrom the mammary fluid. A wide variety of such sample collection devicesare disclosed herein, or are otherwise widely known or described in theliterature, which can be readily adapted for use within specificembodiments of the invention. These collection devices may be providedas a component of the breast pump (such as a removable nitrocellulosefilter placed within the pump, optionally coupled there with by apermanent or removable support member, to directly receive or contactthe expressed mammary fluid as it is pumped), or may be providedseparately (for example as a non-integral membrane, filter, affinitycolumn or blotting material to which mammary fluid or mammary fluidcomponents are exposed to collect a biological sample for assaypurposes). In more detailed aspects, the collection device includes aremovable, fluid-retaining reservoir as described herein below.

In certain embodiments of the invention, kits include reagents and/ordevices for detecting the presence and/or amount of a breast diseasemarker in the biological sample, for example an immunological ormolecular probe that binds or reacts with a breast cancer marker. Amongthese possible reagents immunological and non-immunological probes fordetecting the presence or amount of a breast cancer marker in thebiological sample. The kits may also contain suitable buffers,preservatives such as protease inhibitors, direct or sandwich-typelabels for labeling the probes, and/or developing reagents for detectinga signal from the label. In one aspect, kits of the present inventioncontain monoclonal antibodies useful for detecting and/or measuring abreast cancer marker in a sample. Such antibodies may be pre-labeled, ormay be detected by binding to a secondary antibody optionally includedin the kit. The antibody reagents may be provided in a separatecontainer, or may be provided in combination in a series of containers.Within yet another aspect of the invention, kits containsequence-specific oligonucleotide primers for detecting polynucleotidemolecules encoding breast cancer marker proteins. Such primers may beprovided in separate containers, or may be provided in combinations ofone or more primer pairs in a series of containers. A broad selection ofother kits are provided within the invention based on general knowledgein the art and on the description herein, including kits that containspecific instructions for carrying out the assays of the invention.

Also provided within the invention are methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid, whichmethods employ a novel breast pump 10 or breast pump adapter 12, asdescribed herein below. These methods include a step of applying thebreast pump to induce breast fluid expression, wherein a solid phasesample collection medium is fluidly connected with the breast pump. Thesolid phase sample collection medium may be integrated within the breastpump or otherwise fluidly connected with the pump, so that an expressedbreast fluid sample contacts the collection medium while the pumpremains applied to the breast.

To practice these aspects of the invention, the breast pump 10 (FIG. 1)and breast pump adapter 12 (FIG. 12) each have fluidly connectedtherewith a solid phase sample collection medium selected from any ofthe solid phase media described herein above. The breast pump may begenerally constructed according to various conventional breast pumpdesigns, for example according to the general design described in U.S.Pat. No. 4,929,229 and U.S. Pat. No. 5,007,899 to Larsson; U.S. Pat. No.5,601,531 to Silver; U.S. Pat. No. 3,786,801 to Sartorius; or U.S. Pat.No. 5,295,957 to Aidaet al.

As with other conventional breast pumps, the breast pump 10 of theinvention includes a breast engaging portion 14 constructed of anon-porous material. The engaging portion is sized and dimensioned toreceive at least a nipple 16 portion of a breast 17 and form a suctionseal therewith. Preferably, the breast engaging portion is sized anddimensioned to receive at least an areolar portion of the breast, andmore preferably a distal quarter to one-half or larger portion of thebreast (e.g., as shown in FIG. 1), and form a suction seal therewith.Different sizes and dimensions of the breast engaging member may beselected, e.g., to receive human breasts of differing sizes.Alternatively, devices for veterinary use are provided wherein thebreast engaging member is sized and dimensioned to receive a breast of anon-human mammal.

To form a suction seal with the breast 17 as described above, the breastengaging portion 14 of the pump 10 may be constructed in a variety ofshapes and dimensions. In one embodiment the engaging portion is formedas a simple cylinder, tube or funnel shaped and dimensioned to engagethe nipple 16 or areolar portion of the breast in a suction seal.Preferably, a terminal edge 18 of the engaging portion is rounded orflared so that the edge does not impinge uncomfortably against the skinof breast 17 when negative pressure is applied to the breast to form thesuction seal. In preferred embodiments the engaging portion is roughlyfunnel shaped to comfortably engage a distal quarter to one-half orlarger portion of the breast, as shown in FIG. 1 and form a suction sealtherewith.

The breast engaging portion 14 of the breast pump 10 can be constructedof any suitable non-porous material which is inert to body fluids andwhich has sufficient rigidity to prevent collapse of the engagingportion when negative pressure is applied against its inner walls 20.Preferably, the engaging portion and other parts of the breast pump areautoclavable for sterilization purposes. Thus, the engaging portion maybe constructed of a rigid material such as a polypropylene,polyurethane, polyvinyl plastic, polymethyl methacrylate, and the like.Alternatively, the engaging portion may be constructed of a semi rigidmaterial which prevents collapse but allows for manual compression of atleast a base 22 of the engaging portion to massage the nipple 16 and/orareolar region of the breast 17 to facilitate breast fluid expression.Suitable materials in this context include rubber or syntheticelastomers, e.g., silicon plastic (silastic) and like materials.Preferably, the material that forms the engaging portion is transparentto allow a physician or technician using the breast pump to visualizethe breast 17 to determine its positioning and condition duringapplication of the pump and to observe fluid expression from the nipple.

The breast engaging portion 14 of the breast pump 10 is fluidlyconnected to a sample collection housing 30 made of a rigid material(preferably transparent plastic). The solid phase sample collectionmedium, as described above, is supported in fluid connection with thebreast engaging portion, for example by anchoring the solid medium to,or within, the breast engaging portion or sample collection housing.Typically, the solid phase sample collection medium is affixed within aninterior compartment or lumen 58 of the sample collection housing orcorresponding, fluidly connected, interior space of the breast engagingportion-using any of a wide range of optional anchoring or positioningmeans. Preferably, the solid phase medium is removably supported influid connection with the breast engaging portion, e.g., by means of acloseable retainer or replaceable cassette (see below).

In one aspect of the invention the sample collection housing 30 orbreast engaging portion 14 supports a sample collection pad, or sheet,38 of absorbent or adsorbent material, for example a membrane 39 orfilter 40 pad or sheet (FIGS. 2-5). Multiple pads or sheets (of the sameor different material) may be used in combination. For example, amembrane 39 (e.g., nitrocellulose) may be supported on a filter 40(e.g., a paper filter) as shown in FIG. 5. In this manner, a first sheetmay serve as a support member, a wetting member, a wicking member, or apartitioning member for a second sheet, or may introduce or remove achemical reagent, probe, blocking agent, buffering agent, denaturingagent, etc. therefrom. In one aspect, the multiple sheet materialspartition components of the breast fluid (e.g., by using differentmaterials to retain different components of the breast fluid), therebyallowing for collection of different samples simultaneously.

In another aspect of the invention the housing 30 or breast engagingportion 14 supports a particulate solid phase sample collection medium41, for example beads, resins, microspheres, particulate chromatographicmedia (e.g., agarose or silicate media), and the like (see, e.g., FIG.9). In yet another aspect of the invention, the housing or breastengaging portion supports a non-particulate solid template for samplecollection, for example one or more capillary tubes 42 (FIG. 6), coatedtubes 43 (FIG. 10), plates, wells, slides and the like formed of glass,plastic or other suitable materials.

As shown in FIGS. 1 and 2, a preferred design of the breast pump 10includes a removable coupling mechanism between the engaging portion 14and the sample collection housing 30. A preferred coupling mechanismincludes complementary threads 44, 46, disposed at mated connecting ends48, 50 of the engaging portion, and housing, respectively.Alternatively, a simple pressure fit coupling may be provided toremovably couple mated connecting ends 48, 50 of the engaging portionand housing, as shown in FIG. 9. In yet another alternative embodiment,the connecting ends 48, 50 are removably coupled by a hinge 52 and latch54 that pivotally connects the two connecting ends (FIG. 10).

The sample collection housing 30 or breast engaging portion 14 cansupport the solid phase sample collection medium in several ways, asexemplified in the drawings and by a variety of equivalent designs andconfigurations that will be apparent to the artisan. In preferredembodiments of the invention, the solid phase medium is held on orwithin a support member 56 adapted to support the solid phase medium influid connection with the breast engaging portion, for example a supportthat is fixedly interposed between the engaging portion 14 and thesample collection housing.

Thus, in one exemplary design shown in FIGS. 1 and 2, the support member56 is a removable disc spanning a lumen 58 of the housing and interposedbetween connecting ends 48, 50 of the engaging portion and housing. Foruse in conjunction with a variety of breast pump designs, a diameter 59(FIG. 3) of the support member is between about ¼-3.0 inches, preferablyabout ½-2.0 inches, and more preferably about ¾-1 inches. In preferredaspects, the disc-shaped support member seats within a circumferentialgroove 60 in the connecting end 48 of the housing. A complementarycircumferential groove 62 in the connecting end 46 of the engagingportion opposes the circumferential groove in the connecting end of thehousing to sandwich the disc-shaped support member therebetween.

In this embodiment, prior to connecting the engaging portion 14 of thebreast pump 30 with the housing 30, the support member is seatedtherebetween (e.g. by fitting the support member within the opposingcircumferential grooves 60, 62 of the housing and engaging portion). Theforce of connection (i.e. threading, pivoting or pushing the engagingportion and housing relative to one another) firmly sandwiches thesupport member in position between the engaging portion and housing.

To facilitate this purpose, the thickness (i.e., sectional height) 63 ofthe support member 56 in the present embodiment is equal to or slightlygreater than the height of a sidewall 64 of the circumferential groove60 of the housing 30, whereby the support member is held in a frictionfit and may be partially compressed when the engaging portion andhousing are connected. Thus, the thickness of the support member isbetween about 2 mm to 5 cm, preferably about 3 mm to 2 cm, and morepreferably about 4 mm to 1 cm. Consistent with this design, the supportmember can be made of a hard plastic material (e.g., a hard polyvinyl orpolyurethane), but is preferably made of a resilient, moderatelycompressible material, e.g., soft plastic, rubber, or a waterproof fiberor composite material as used in conventional plumbing and automotivegaskets.

A disc-shaped support member 56 is well suited to support a sheet 38 ofabsorbent or adsorbent material, such as a membrane or filter. As shownin FIGS. 2 and 7, the sheet is preferably sandwiched between an upperretainer ring 66 and a lower retainer ring 68 of the support member tohold the sheet in place against negative pressure that may pass throughthe filter when a vacuum is applied through the engaging portion 14 andhousing 30 (see below), as well as when the nipple 16 impinges againstthe sheet. The upper and lower retainer rings may be integrally joinedin a disposable refill as shown in FIG. 2, or the two retainer rings maybe separable to provide a reusable cassette for removing and insertingreplacement sheets. An example of the latter design is depicted in FIG.7, where the upper and lower retainer rings are releasablyinterconnected, e.g., by a hinge 71 or other connecting means such as aninterlocking threading or detent fit mechanism. In this embodiment theupper and lower rings can be opened or disconnected to allow insertionand removal of the sheet, and juxtaposingly closed, e.g., by a snap 72on one ring adapted to form a detent fit within a receptacle 74 on theopposing ring, thereby holding the sheet in a fixed position between thetwo rings. To facilitate this purpose, opposing faces 75 of the upperand lower rings may have a rugose or otherwise decorated surface tofacilitate retention of the sheet, for example a ridge 76 or ridges toengage the sheet and securely clamp the sheet between the tworings.

In an alternative design depicted in FIGS. 4 and 5, there is no upperretaining ring 72 and the sheet 38 simply rests upon the support member56 or is removably retained against an upper surface 76 of the supportmember by alternative retaining means. For example, the sheet may befitted within a recess 78 surrounding the upper surface of the supportmember that is shaped and dimensioned to receive the sheet. The sheetmay be securely fitted within the recess, e.g., by appropriately sizingthe sheet so an edge of the sheet frictionally engages a sidewall 79 ofthe recess. Alternatively, a retaining groove may be provided betweenthe sidewall of the recess and the upper surface of the support memberto receive the edge of the sheet and thereby retain the sheet by adetention fit within the recess during use. In yet another alternativedesign, the sheet simply rests atop the upper surface of the supportmember and is removably secured thereto, e.g., by wetting or gluing(preferably with an inert bonding agent) to create a temporary bondbetween the sheet and upper support member surface. In each of theforegoing designs, the sheet can be easily seated within or atop thehousing for sample collection and removed thereafter for processing,e.g., by hand or using forceps or other conventional handling tools.

In preferred embodiments of the invention, the support member 56includes a recess 78 which forms a fluid-retaining well, as shown inFIG. 5. The recess can thus be filled with a desired solution, such as abuffer, a solution containing a probe, cross-linking agent, blockingagent, denaturing agent, etc., to facilitate sample collection,handling, and/or processing.

Where the design of the support member 56 is such that it spans thelumen 58 of the sample collection housing 30 or corresponding interiorcompartment of the breast engaging member 14, or when the support membercontains a recess 78 forming a well, it is generally desirable toprovide air channels 80 in the support member 56 to allow negativevacuum pressure to pass from the housing through the air channels to theengaging portion 14 of the pump during operation, and to allow ventingof the engaging portion and housing to permit disengagement of theengaging portion from the breast 17 after use. Preferably, one or moresuch air channels are located near the periphery of the support member,as shown in FIGS. 2, 3, 5, 6 and 9. Alternatively, one or more airchannels may be centrally located, as shown in FIG. 4. The air channelsmay be positioned so that they do not communicate with the solid phasesample collection medium, as shown in FIGS. 2, 3, 5, 6 and 9, or theymay communicate and form a gaseous connection therewith (provided thatthe solid phase medium is porous and has sufficient strength towithstand vacuum pressures transmitted through the air channel), asshown in FIG. 4.

Alternative designs and configurations of the housing 30, breastengaging member 14, and/or support member 56 are also provided whichvary with the type of solid phase sample collection medium used. Forexample, when a particulate solid phase sample collection medium 41(e.g. beads, resins, or microspheres) is used, the medium may beenclosed in a cartridge 82 removably mounted to, or integrated within,the support member or otherwise removably connected to the samplecollection housing 30 or breast engaging portion. As shown in FIG. 9,preferred embodiments of the invention provide a removable engagementmechanism which allows the cartridge or other receptacle containing thesolid phase medium to be removably engaged relative to the housing orbreast engaging member, e.g., by engaging the cartridge with a supportmember so that a first end of the cartridge makes a fluid connectionwith the engaging portion 14 of the pump 10. In one embodiment, thefirst end of the cartridge is removably inserted through a mountingchannel 86 that passes through the support member to provide a fluidconnection between the engaging portion of the pump and the cartridgefirst end. Preferably, the channel is dimensioned to receive the firstend of the cartridge in a friction fit (e.g., wherein a diameter of thechannel is about 0.5 mm to 2 cm, preferably about 1 mm to 1 cm, and morepreferably about 3-5 mm), whereby the cartridge can simply be pushedinto the channel until the cartridge first end is flush with, or extendsslightly above, the upper surface 76 of the support member and willremain in place during use. For this purpose it is also preferable toform at least the channel portion of the support member from aresilient, moderately compressible material so that the channelyieldingly receives and releases the cartridge in a moderate (i.e.,readily hand removed) friction fit. Alternatively, the cartridge can beengaged relative to the housing by complementary threading orinterlocking detent fitting (e.g. a conventional key and groove design)between the cartridge first end and the support member channel). In yetother alternative designs the cartridge can be permanently engaged withthe support member or engaged directly to the housing.

Design and construction of the cartridge 82 will vary depending on thecharacteristics of the particulate solid phase medium used, includingthe size of the particles, the function of the particles (e.g.,chromatography adsorption, affinity binding, etc.), and whether theparticles are used dry or are contained in a solution, among otherfactors. Design and construction of the cartridge will further depend onthe type of breast diseasemarker(s) which may be sought for detection inthe sample (e.g., cells, proteins, lipids or nucleic acids).

In a preferred embodiment shown in FIG. 9, the cartridge is cylindricaland contains beads or microspheres. To enclose the beads or microspheresin the cylinder while maintaining a fluid connection with the engagingportion 14 of the pump 10, the first end 84 of the cylinder is coveredby a semi-permeable cover 90 of a porous barrier material (e.g, a filteror membrane) which allows breast fluid (including or excluding selectedcomponents of the fluid, such as cells) to pass through the cover tocontact the beads or microspheres, while preventing escape of the beadsor microspheres from the cartridge. In this manner, the cover canpartition components of the breast fluid into the cartridge, and canalso separately retain different components on the cover, therebyallowing for collection of different samples simultaneously. Thesemi-permeable cover can be affixed to the cartridge by a variety ofmeans, e.g., by bonding with a removable or permanent bonding agent, orby providing a removable or integral cover retaining ring 92 to securethe cover to the cartridge first end 84. A second end of the cartridgefeatures a second end cover 96 which may be integral to or removablefrom the cartridge, and which may be impermeable to gas and fluids orsemi permeable as described above for the first end cover.

In another aspect of the invention, the housing 30 supports anon-particulate solid template for sample collection. This type of solidphase collection medium includes, e.g., one or more capillary tubes 42(FIG. 6), coated tubes 43 (FIG. 10), plates, wells, slides and the like.These templates for receiving, adsorbing or binding a sample of breastfluid (or desired components thereof) are preferably formed of glass,plastic or like materials known in the art to be suitable for samplecollection (e.g. inert plastics).

To accommodate these various templates, yet additional alternativedesigns and configurations of the housing 30, breast engaging portion14, and/or support member 56 are provided. For example, when capillarytubes 42 are used, these may be mounted to or integrated within thesupport member, or anchored by a variety of other comparable means withrespect to the housing 30. As shown in FIG. 6, preferred embodiments ofthe invention utilize a support member with one or more mountingchannels 86 to removably receive a first end of one or more capillarytubes 42, so that the end of the tube makes a fluid connection with theengaging portion 14 of the pump 10. Thus, the channels have a preferreddiameter equal to or slightly less than a diameter of a standardcapillary tube, i.e., about 0.5 mm to 3 mm, preferably about 1-2 mm andmore preferably about 1.5 mm. Construction of the support member andmounting of the tube(s) is similar to support member construction andmounting of the cartridge as described above. When a single tube isused, it is preferably placed centrally relative to the housing. Whenmultiple tubes are used they may be arrayed to collect multiple samplessimultaneously, e.g., as shown in FIG. 6.

Another alternative solid template for sample collection provided withinthe invention is a coated tube 43 which is preferably mounted relativeto the housing 30 in the same manner as described above for capillarytubes 42 (FIG. 10). The tube may be open at both ends, or may have asemi-permeable cover at one or both ends, as well as an impermeablesecond end cover, as described above for the cartridge 82.

The coated tube has a lumenal coating 100 adapted for adsorbing,binding, partitioning or otherwise processing the breast fluid sample.For example, the coating may be an affinity coating having an antibody,ligand, or other binding partner that specifically binds a selectedbreast disease marker, wherein the coating is covalently or otherwisebound to a lumenal wall of the tube. A wide variety of useful coatingsare disclosed herein or are otherwise well known in the art. Thesecoatings may also be used to coat other solid phase media for use withinthe invention, including templates such as wells, plates, slides, etc,including a well formed by a recess 78 in a support member 56.

Because only small droplets of breast fluid will typically be expressedat the surface of the nipple 16, it is generally preferred to directlycontact the expressed fluid on the nipple with the solid phase samplecollection medium. This requires positioning of the sample collectionmedium close to the base 22 of the breast engaging portion 14 of thepump as shown in the figures. Thus, when a support member 56 is providedit is positioned so that its upper surface 76 will directly contact thenipple during application of negative pressure through the engagingportion to the breast. Only approximate positioning is generallyrequired in this regard, because the nipple will tend to be drawn towardthe support member by the vacuum and thereby will abut the upper supportmember surface.

However, because breast pump designs and breast anatomy varysignificantly, it is preferable to adjustably mount the solid phasemedium relative to the housing 30 so that it can be moved closer to, orfarther away from, the base 22 of the engaging portion 14 of the pump10. Thus, in preferred embodiments of the invention a reciprocatingmechanism is provided which adjustably moves the solid phase collectionmedium in closer, or more distant, proximity to the nipple when thebreast pump is engaged therewith. At the beginning of the fluidexpression procedure, the collection medium is retracted away from thenipple while negative pressure is applied to the breast to facilitatefluid expression. Fluid expression is visualized through a transparentengaging portion or housing, and the collection medium is then advancedproximal to the nipple to contact the expressed fluid.

As shown in FIG. 11, a preferred design for the reciprocating mechanismincorporates a support member 56 to support the solid phase collectionmedium, as described above. The support member is reciprocatinglymounted relative to a rotating member 109 of the housing 30, preferablyon a reciprocating carrier 110. The support member may be removablymounted to the carrier, e.g., by friction fitting, detention fitting orthreadedly engaging the support member to a first end 112 of thecarrier, as described above for mounting the support member to thehousing and/or engaging portion 14 of the pump 10. For example, thesupport member may be mounted by friction fitting within acircumferential groove 114 at the first end of the carrier. Inconjunction with this design, the carrier is preferably in the form ofan open cylinder so that negative pressure can be effectivelytransmitted through the carrier and support member to the engagingportion.

To reciprocatingly adjust the position of the carrier 110 and/or supportmember 56 relative to the engaging portion 14 of the pump 10, therotating member 109 of the housing 30 is sealably, rotatably, andremovably interconnected to the base 22 of the engaging portion. Thisinterconnection may be accomplished by a variety of designs, one ofwhich is to seat a first O-ring 116 in opposing circumferential grooves118, 120 in the connecting ends 48, 50 of the engaging portion, and therotating member of the housing, respectively. These grooves are sizedand dimensioned to receive the O-ring in an airtight seal when vacuumpressure is applied through the housing and engaging portion of thepump, without substantially compressing the O-ring. The O-ring is alsolubricated, e.g, with silicon grease. These features allow free rotationof the rotating member of the housing relative to the engaging portionof the pump, which rotation drives the reciprocating mechanism toadvance the sample collection medium (e.g., by advancing the carrierand/or support member) to contact the expressed breast fluid on thenipple 16.

To complete the reciprocating mechanism for the above describedembodiment of the invention, the rotating member 109 of the housing 30is also sealably and rotatably interconnected to a stationary member 124of the housing. This interconnection is preferably achieved by seating asecond O-ring 126 in opposing circumferential grooves 128, 130 in a rearconnecting end 132 of the rotating member of the housing and a frontconnecting end 134 of the stationary member 124 of the housing,respectively. These grooves are also sized and dimensioned to receivethe O-ring in an airtight seal without substantially compressing theO-ring, and the O-ring is lubricated to facilitate free rotation of therotating member relative to the stationary member.

To reciprocate the carrier 110 and/or support member 56 forward andbackward relative to the engaging portion 14, the rotating member 109 ofthe housing 30 is provided with a lumenal, helically oriented groove 140dimensioned to receive a riding peg 142 extending transversely from thecarrier or support member. In addition, the rotating member of thehousing is provided with a longitudinally oriented, lumenal groove 144dimensioned to receive an angularly fixating keel 146 extendingtransversely from the carrier or support member. In accordance with thisdesign, rotation of the rotating member 109 of the housing 30 drivesrotation of the carrier or support member which is angularly fixedrelative to the rotating member by the fixating keel engaged with thelongitudinal groove of the rotating member. As the rotating member ofthe housing and carrier thus rotate (with the position of the engagingportion and stationary member of the housing angularly fixed by frictionor manual or structural resistance), the riding peg rides along thehelical groove, translating the peg in the direction of the groove andthereby causing the support member or carrier to reciprocate forward orbackward relative to the engaging portion.

To insert and remove the solid phase medium and/or support member 56from the rotating member 109 of the housing 30, a removableinterconnection is provided between the rotating member and the base 22of the engaging portion, as described above. To uncouple the rotatingmember and engaging portion, all that is required is that these parts bepulled in opposing directions, whereby the O-ring 116 will unseat fromone of the opposing circumferential grooves 118, 120 in the connectingends 48, 50 of the engaging portion and rotating member, respectively.To recouple the rotating member and engaging portion after loading orretrieval of the sample collection medium and/or support member, theyare simply pushed back together. To facilitate reseating of the O-ring,it may be desired to make one of the opposing circumferential groovesdeeper than the other, so that the deeper groove retains the O-ring whenthe rotating member and engaging portion are separated, and theshallower groove more readily accepts the O-ring when they arere-coupled.

An alternative reciprocating mechanism is provided within the inventionwhich uses a simple slide mechanism to reciprocate the sample collectionmedium relative to the engaging portion 14 of the pump 10, as shown inFIGS. 14 and 15. One embodiment of the slide mechanism features amanifold 150 defining an inner lumen 152 that is not in gaseousconnection with an outer lumen 154 of the housing. This design providesfor a manual slide lever 156 to extend to the outside of the housing sothat a head portion 158 of the lever can be manually engaged by a pumpoperator. The slide lever is in turn connected to the support member 56or carrier 110 which are sized and dimensioned to allow the carrier toreciprocate freely within the inner lumen.

In operation, the slide lever 156 is moved to a rearward position sothat the solid phase sample collection medium (e.g., a pad or sheet 38of absorbent material is out of contact with the nipple 16, as shown inFIG. 14. Negative pressure is applied through the outer lumen 154 to thearea of the breast surrounding the nipple, the tip of which is alignedwith the inner lumen. Breast fluid expression is visualized through thetransparent engaging portion and housing, at which time the lever ismanually engaged by the head portion 158 and moved forward. Movement ofthe lever causes the support member and/or carrier to move forward untilthe sample collection medium contacts the expressed fluid at the tip ofthe nipple. The engaging portion and housing are removably connected,e.g., by a hinge 52 and latch 54 or other suitable connection means,thereby allowing for easy insertion and removal of the solid phasemedium and/or support member.

In each of the foregoing breast pump designs, the engaging portion 14 ofthe breast pump 10 is in gaseous connection with a vacuum pump 160capable of generating sustained negative pressure in an area of thebreast 17 surrounding the nipple 16 (see FIG. 1). Any of a large varietyof vacuum pumps, which are well known for use in conjunction with breastpumps, can be used, including manual pumps (FIG. 1), mechanically drivenpumps and electrically driven pumps. When activated, the pump generatesnegative pressures of between about 50-200 mm Hg. Typically the pumpwill be connected via a heavy vacuum hose 162 in connection with theengaging portion. Generally, the hose is connected to the housing 30which will is in gaseous connection with the engaging portion (see, e.g,FIGS. 1, 8 and 11).

Pressure exerted upon the breast 17 by the pump can be varied inaccordance with well known pressure modulating mechanisms (e.g., byproviding a diaphragm or other mechanism to modulate a diameter of an inline, pressure modulating valve). In addition, the breast pump 10includes a venting mechanism, e.g., a pressure release valve 164, whichthe user can selectively operate to close and vent the system before andafter use, thereby selectively applying and releasing the vacuumpressure acting on the breast. In this regard, the system is generallyvented as soon as sufficient breast fluid expression is observed by theoperator. This also relieves pressure on seals (e.g., O-rings 116, 126),when the reciprocating mechanism relies on a sealable and rotatableconnection between different parts of the pump (as in FIGS. 11 and 13),thereby facilitating respective rotation of the different parts toreciprocate the support member 56 and/or carrier 110.

In yet another aspect of the invention, a breast pump adapter 12 isprovided which couples a solid phase sample collection medium with aconventional breast pump (See FIGS. 12 and 13). As shown in FIG. 12, theadapter features a replacement breast engaging portion 170 sized anddimensioned for removable insertion within a breast engaging portion 14of a conventional breast pump. In preferred embodiments, the replacementbreast engaging portion is funnel shaped and nests within a funnelshaped breast engaging portion of an existing breast pump. When fullynested, a terminal edge 172 of the replacement breast engaging portionextends at least as far as the terminal edge 18 of the breast engagingportion of the existing breast pump.

The replacement breast engaging portion 170 can be removably connectedto the breast engaging portion 14 of the existing breast pump 10 by avariety of means, e.g, by friction fitting, detention fitting orthreadedly engaging the replacement engaging portion with the breastengaging portion of the existing pump. Preferably, the adapter 12 has astem portion 174 that extends into a cylindrical, connecting portion 176of the existing pump, and the stem portion cooperates with this part ofthe existing pump to provide a removable connection mechanism. Thus, inone preferred embodiment the stem portion features a circumferentialgroove 178 dimensioned to receive an O-ring 180, which O-ring impingesagainst an inner wall 182 of the connecting portion to create a frictionfit to interconnect the replacement engaging portion with the breastengaging portion of the existing pump.

The adapter 12 supports a solid phase sample collection medium in fluidconnection with the replacement engaging portion 170. Preferably, thesolid phase medium is connected with the replacement engaging portion bya support member 56, as described above. The support member may beintegrally or removably mounted to the adapter, e.g., by frictionfitting, detention fitting or threadedly engaging the support member tothe stem 174 of the replacement engaging portion, in a position thatwill allow contact between the nipple and solid phase medium during orafter breast fluid expression. For example, the support member may bemounted by friction fitting within a circumferential groove 184 at abase of the stem (FIG. 12).

In preferred embodiments of the breast pump adapter 12, a reciprocatingmechanism is provided to move the solid phase sample collection mediumrelative to the replacement engaging portion 172, in accordance with theconcepts described above. As shown in FIG. 13, a preferred design forthe adapter having a reciprocating mechanism features a replacementbreast engaging portion 170 sealably and rotatably nested within arotating dial member 190, which is in turn sized and dimensioned forremovable insertion within a breast engaging portion 14 of an existingbreast pump. The replacement breast engaging portion and rotating dialmember are preferably funnel shaped to collectively nest within a funnelshaped breast engaging portion of a conventional breast pump. When fullynested, a terminal edge 172 of the replacement breast engaging portionand free edge 192 of the rotating dial member extend at least as far asthe terminal edge 18 of the breast engaging portion of the existingbreast pump.

The rotating dial member 172 is connected to a rotating member 109 ofthe housing 30, preferably as a unitary insert, whereby manual rotationof the dial member drives rotation of the rotating member of thehousing. The rotating member of the housing is in turn rotatably coupledwith an anchoring member 194 of the housing which anchors the entirehousing within the existing pump, e.g., within a cylindrical, connectingportion 176 of the existing pump. As shown in FIG. 13, the anchoringmember of the housing is preferably in the form of a sleeve thatpartially surrounds the rotating member of the housing and is sealably,rotatably connected therewith. The anchoring member is in turnnon-rotatingly anchored within the cylindrical, connecting portion ofthe existing pump.

In one preferred embodiment the rotating member 109 of the housing issealably, rotatably connected with the anchoring member 194 of thehousing by seating a first O-ring 196 in opposing circumferentialgrooves 198, 200 at front connecting ends 202, 204 of the rotatingmember and the anchoring member 194 of the housing, respectively. Thesegrooves are sized and dimensioned to receive the O-ring in an airtightseal between the rotating member and anchoring member, withoutsubstantially compressing the O-ring. The O-ring is also lubricated tofacilitate free rotation of the rotating member relative to theanchoring member. A second, lubricated and non-compressingly seatedO-ring 206 is seated in opposing circumferential grooves 208, 210 atrear connecting ends 212, 214 of the rotating member and anchoringmember of the housing, respectively, to facilitate rotation of therotating member relative to the anchoring member.

To align and facilitate rotation of the rotating member 109 of thehousing, the rotating dial member 190 (which drives the rotating memberof the housing) is sealably, rotatably connected with the replacementengaging portion 170 of the adapter 12. Preferably, the replacementengaging portion has a stem 216 which nests within a stem-shaped base218 of the rotating dial member. Free rotation between these structuresis achieved, e.g., by providing a third lubricated and non-compressinglyseated O-ring 220 seated in opposing circumferential grooves 222, 224 inthe stem and base of the replacement engaging portion and rotating dialmember, respectively. This rotation is also facilitated by frictioncontact (by pressure and/or suction) between the replacement engagingportion and the breast 17 of the patient, which angularly secures thereplacement engaging portion and prevents its co-rotation with therotating dial member.

The anchoring member 194 of the housing is in turn anchored within theexisting pump by an anchoring mechanism that angularly secures theanchoring member within the pump, e.g., against an inner wall 182 of thecylindrical connecting portion 176. For example, front and rearcompressible anchoring sleeves 230, 232 may be mounted in front and rearcircumferential anchoring sleeve retainer grooves 234, 236 surroundingthe anchoring member. The anchoring sleeves are non-lubricated and aremade of a semi-compressible material such as rubber or soft plastic.This construction creates a friction anchor between the anchoring memberand the inner wall of the connecting portion, so that the anchoringmember does not move angularly during rotation of the rotating member109 of the housing. Both the anchoring sleeves and retainer grooves arepreferably sharply angled at a position corresponding to the bases ofthe retainer grooves (i.e., they have a rectangular or triangularcross-section), to securely retain the anchoring sleeves in the groovesdespite strong friction against the inner wall of the connecting portionwhen the anchoring member of the housing is being inserted into theconnecting portion of the existing breast pump 10 to assemble theadapter 12 with the pump.

Because the replacement engaging portion 170 is anchored by frictionagainst the breast 17, and the anchoring member 194 of the housing 30 isanchored by friction against the inner wall 182 of the connectingportion 176 of the existing pump 10, the rotating member 109 of thehousing rotates freely with respect to both the replacement engagingportion and the anchoring member when an operator manually engages therotating dial member 190 and turns it gently while maintaining pressureagainst the breast.

Relative rotation between the rotating member and anchoring member ofthe housing drives the reciprocating mechanism within the instantembodiment of the invention to advance the sample collection medium(e.g., by advancing a carrier 110 and/or support member 56 supportingthe medium) toward the replacement engaging portion 170 to contact theexpressed breast fluid on the nipple 16. As with previously describedembodiments, the housing 30 preferably houses a support member 56 tosupport the solid phase collection medium, as described above. Thesupport member is reciprocatingly mounted relative to the anchoringmember 194 of the housing 30, preferably on a reciprocating carrier 110.The support member may be removably mounted to the carrier, e.g., byfriction fitting, detention fitting or threadedly engaging the supportmember to a first end 112 of the carrier, as described above. In theembodiment shown in FIG. 13, the support member is removably engagedwith the carrier by cooperative threading 140 between the support memberand carrier. In addition, the support member may be sized anddimensioned for receipt within the stem 216 of the replacement engagingportion, because the replacement engaging portion and an inner (i.e.,lumenal) diameter of the stem thereof are smaller than respectivedimensions of the original engaging portion 14 and its base 22, so thatthe nipple may not fully extend through the stem to contact thecollection medium within the housing. Also in conjunction with thisdesign, the carrier is preferably in the form of an open cylinder andthe rotating member 109 of the housing has a vacuum port 242 so thatnegative pressure can be effectively transmitted through the rotatingmember and carrier (and/or through air channels 80 of the supportmember) to the replacement engaging portion.

To reciprocatingly adjust the position of the carrier 110 and/or supportmember 56 relative to the replacement engaging portion 170 of theadapter 12, the anchoring member 194 of the housing is provided with alumenal, helically oriented groove 140 dimensioned to receive a ridingpeg 142 extending transversely from the carrier or support member. Inaddition, the rotating member of the housing is provided with alongitudinally oriented, lumenal groove 144 dimensioned to receive anangularly fixating keel 146 extending transversely from the carrier orsupport member. Lastly, the rotating member is provided with a second,longitudinally oriented, lumenal groove 244 to allow access of theriding peg through the wall of the rotating member of the housing intothe helically oriented groove and to allow reciprocating passage of thepin along the groove.

In accordance with this design, rotation of the rotating dial member 190drives rotation of both the rotating member 109 of the housing 30 aswell as the carrier 110 (or support member) which is angularly fixedrelative to the rotating member by the fixating keel 146 engaged withthe longitudinal groove 144 of the rotating member. As the rotatingmember and carrier thus rotate (with the position of the replacementengaging portion 170 and anchoring member 194 angularly fixed byfriction or manual or structural resistance), the riding peg rides alongthe helical groove 140, translating the peg in the direction of thegroove and thereby causing the support member or carrier to reciprocateforward or backward relative to the replacement engaging portion.

To insert and remove the solid phase medium and/or support member 56from the adapter 12, removable connections can be uncoupled between theexisting pump 10 and the entire adapter unit, between the rotatingmember 190 and anchoring member 194 of the housing, or between therotating dial member and replacement engaging portion 170, among otheraccess designs which will be readily apparent to those skilled in theart.

In more detailed aspects of the invention illustrated in FIGS. 16-26,selected features of a general purpose breast pump 10 for mammary fluidsample collection as described above are incorporated within a hand-heldbreast pump device 10′—adapted for greater fidelity and ease of samplecollection. The hand-held sample collection pump is uniquely designedand constructed to incorporate the breast engaging member 14 and thevacuum pump mechanism 160 in a compact, structurally integrated breastfluid collection apparatus that can be manipulated and operated with onehand. As with the general-purpose breast pump 10, described above, thehandheld pump 10′ incorporates the vacuum pump mechanism 160 in gaseousconnection with the breast engaging element 14 to route suction pressurefrom the pump mechanism, through the engaging element, to apply negativepressure in the area of the nipple 16 of the patient. The solid phasesample collection medium (e.g., a membrane, filter, particulate medium,and/or a non-particulate solid collection template such as a plastic orglass tube, well, vial or slide) is likewise fluidly connected with thebreast engaging member—to provide for direct or indirect transmission ofthe expressed breast fluid through the engaging member to contact thesolid phase collection medium.

According to these aspects of the invention, novel breast fluid samplecollection methods are provided wherein a doctor, technician or patientcollecting a breast fluid specimen can grasp and operate the hand-heldbreast pump 10′ to stimulate expression of the breast fluid and collecta specimen thereof while keeping one hand free for additional tasks,such as monitoring the patient and recording patient information. Inthis regard, the compact pump design allows the device to be picked upand manipulated with one hand-to seat the breast engaging elementagainst the breast of the patient and thereafter apply vacuum pressureto the breast by manual operation of the vacuum pump 160 to stimulateexpression of breast fluid. This action causes a suitable volume ofbreast fluid to be expressed at or near the nipple 16 for samplecollection. In conjunction with these simple operation steps, thehand-held device also allows for simultaneous collection of theexpressed breast fluid onto, or within, the solid phase samplecollection medium that is fluidly connected with the engaging member,often without additional manual steps or a need to remove the devicefrom the breast or otherwise engage two hands in the operation.

The hand-held breast pump device 10′ can be employed for collection ofbreast fluid following oxytocin stimulation to facilitate breast fluidexpression, as described above. Alternatively, the device can be usedwithout oxytocin priming to achieve breast fluid expression by vacuumpressure alone, optionally coupled with mechanical breast stimulation,in a substantial percentage of subjects.

In using the hand-held breast pump 10′ of the invention expressed breastfluid is typically transferred directly upon expression to the solidphase sample collection medium, without intervening manual steps or arequirement to remove the breast engaging member 14 from the breast 17before the sample is collected. Sample collection in this manner israpid and simple, and promotes sanitary application of the device tominimize the risk of patient infection and sample contamination. Thus,within certain methods of use for the hand-held breast pump, breastfluid is directly transferred to a solid phase collection medium, forexample a membrane, filter, reservoir or vial, integrated within thehand-held pump. The sample may be collected as whole, undiluted breastfluid containing constituent proteins, particulates and/or cells.Alternatively, selected components of the expressed breast fluid may besimultaneously or subsequently removed from the fluid (e.g., byfiltering, partitioning, or refining the breast fluid) to yield aprocessed fluid sample and/or to obtain a solid phase constituentsample. For example, various collection methods are provided which yieldseparated solid components (e.g., cells or other particulates) from thefluid. Alternate collection methods yield soluble, suspended, or solidphase captured proteins, lipids, carbohydrates, polynucleotides or othermolecular/biochemical components from the expressed breast fluid. Incertain embodiments, the hand-held pump device functions to separate orpartition a desired protein, lipid, carbohydrate, or polynucleotidesample material into a solid phase collection medium, such as amembrane, filter, or chromatographic substrate (e.g., leptin-,antibody-, enzyme-, or ligand-coated vials, beads, etc.) In this regard,various alternative or additional steps from knowncollection/chromatographic methods can be employed during samplecollection and processing according to the invention. In this manner,proteins, lipids, carbohydrates, polynucleotides, cells, and noncellularparticulates may be partitioned from liquid components of the breastfluid, or separated from one another, simultaneous with or subsequent toexpression of the fluid, by various known methods, including membraneadsorption, filtering, affinity chromatography, chemical processing,centrifugation, etc. to yield a range of constituent or processedsamples.

In certain collection methods of the invention, breast fluid expressedby use of the hand-held pump 10′ is simultaneously or subsequentlydiluted, filtered, washed, admixed with fixative or other processingagents, or otherwise processed or modified to yield a collected fluidsample partially or completely devoid of cells, proteins and/or otherselected components originally present in the expressed fluid, toprovide a processed fluid sample for laboratory analysis. In otherembodiments, particulate components of the breast fluid, for example,cells, cellular components and/or cellular debris, are collected afterprocessing and/or modification, e.g., for cytological examination. Theseand other alternative collection methods involving preliminary sampleprocessing in conjunction with use of the hand-held breast pump 10′ areoptionally performed simultaneous with, or closely following expressionof the breast fluid. Often, sample collection is coincident with thefluid contacting one or more solid phase collection medium(a) fluidlyconnected with the breast engaging member 14. Depending on the type(s)of medium(a) used, preliminary sample processing can also be achieveddirectly by simple operation of the pump, without the need foradditional processing steps or removal of the breast engaging member 14from the subject's breast. For example, the expressed breast fluid maybe sequentially transferred through multiple media, e.g, through afilter or membrane into a liquid-retaining reservoir or container,thereby separating certain components for further processing oranalysis. Alternatively, the expressed fluid may be partially processedcoincident with transfer to the solid phase medium(a) by chemical orphysical reaction (e.g., adsorption, covalent or affinity linkage,enzymatic reaction, etc.) with the medium or a coating or secondaryprocessing agent admixed or linked therewith.

In yet other alternative methods within the invention, preliminarysample processing involves additional steps following breast fluidexpression. In certain embodiments, the breast engaging member 14 isremoved from the breast after the breast fluid is expressed and thefluid is transferred to a first solid phase sample collection medium,typically a membrane or filter. This initial or primary stage of samplecollection may be followed by washing or by manual transfer of selectedbreast fluid components (e.g., proteins, carbohydrates, cells, orcellular debris) from the first solid phase collection medium (e.g., anitrocellulose membrane 39) to a second solid phase medium, e.g., aglass slide or fluid-containing reservoir. Typically, preliminary sampleprocessing in this regard precedes final packaging of the collectedsample for storage or shipment to a lab for further processing andanalysis of the sample.

In more detailed embodiments of the invention, cells or other cellularmaterials useful for cytological examination are separated orpartitioned simultaneous with or sequential to breast fluid expressiononto or within a first solid phase collection medium. In one example,whole cells are separated from the expressed fluid onto a nitrocellulosemembrane 39 or a filter 40, which is typically secured in fluidconnection with the breast engaging member 14 by a fixed or removablesupport member 56 mounted to the engaging member or sample collectionhousing 30 or otherwise integrated with the hand-held breast pump 10′.The cells are subsequently transferred or washed in fluid (e.g.,cytology fluid) to a second solid phase sample collection medium (e.g.,a slide, well, tube or vial), which may also be connected to, orintegrated with, the breast engaging member or sample collection housingas described.

To facilitate sample collection according to the foregoing embodimentsof the invention, the hand-held breast pump 10′ is typically provided asa compact, hand-held unit for ease of use and convenience of storage. Asdepicted in FIG. 16, certain embodiments of the hand-held pump comprisea modular device formed of a plurality of components that are joined orsecurable in fixed structural interconnection with one another. Thesecomponents, which include a breast engaging member 14, vacuum pump 160and solid phase sample collection component(s), may be partially orcompletely disassembled to remove or uncouple the individual components,or parts thereof, as desired for efficient operation, cleaning,servicing and/or storage.

As described above for the general-purpose breast pump 10 of theinvention, the breast engaging portion 14 of the hand-held pump 10′ isconstructed of a rigid or semi-rigid, non-porous material and is sizedand dimensioned to receive at least the nipple 16 of the subject'sbreast 17 and form a suction seal therewith (see, e.g., FIG. 16). Thebreast engaging portion may be constructed in a variety of shapes anddimensions to accommodate variations in breast anatomy. As alsodescribed above, the terminal edge 18 of the engaging portion is roundedor flared so that the edge impinges comfortably and forms an effectivesuction seal against the skin when negative pressure is applied to thebreast. Typically, the engaging member is constructed of a rigid plasticmaterial which is transparent to allow the operator to visualize thebreast, determine positioning of the device, and observe expression offluid from the nipple 16. Preferably, the engaging portion and otherreusable components of the pump are autoclavable for sterilizationpurposes.

In more detailed embodiments of the invention shown in FIGS. 16-18, thehand-held breast pump 10′ is a modular device comprised of multiple,integrated components that are fixedly joined to one another when thepump is assembled, but can be readily detached or uncoupled from oneanother. This modular configuration of the device allows forinterchanging of parts to adapt the pump for different patients andcollection modes, and to facilitate storage, cleaning and/or servicingof the device. Thus, in one embodiment, the breast engaging portion 14of the pump is provided as a separate, funnel shaped component that isdetachable from one or more interconnecting components of the device(see, e.g., FIG. 18). In this manner, the engaging member can be removedfrom the rest of the device for cleaning and sterilization, or to allowfor interchanging of different engaging members to accommodate breastanatomy differences among patients. Typically, the breast engagingmember is removably coupled with a surface or member of the samplecollection housing 30.

As illustrated in FIG. 18, the hand-held breast pump 10′ of theinvention incorporates the solid phase sample collection medium in fluidconnection with the breast engaging member 14, typically by use of asupport member 56 affixed to, or removably connected with, the engagingmember. In selected embodiments, the support member encloses or supportsone or more pads or sheets of absorbent or adsorbent material, forexample a nitrocellulose membrane 39. Multiple pads or sheets of thesame or different material may be used in combination (e.g., including awetting member, a wicking member, or a partitioning member).Alternatively, the support member can incorporate or support aparticulate solid phase sample collection medium, for example beads,resins, microspheres, particulate chromatographic media (e.g., agaroseor silicate media), and the like. In yet additional aspects of theinvention, the support member engages or supports a non-particulatesolid template for sample collection, for example one or more capillarytubes, coated tubes, plates, wells, slides and the like formed of glass,plastic or other suitable materials. In certain embodiments, the supportmember may incorporate a compartment, well or reservoir to receive orintroduce sample processing agents selected from chemical reagents,probes, blocking agents, buffering agents, denaturing agents.

The support member 56 for use in conjunction with the hand-held breastpump 10′ is typically provided as a removable cassette that can beinserted within the engaging member 14, often to seat against the innerwall 20 thereof (see, e.g., FIGS. 16 and 18. In preferred aspects, thesupport member seats by a friction or compression fit against the innerwall of the engaging member, which may be facilitated by a complementarycircumferential ridge and groove design between the support member andengaging member inner wall, as shown in FIG. 16.

In certain embodiments of the hand-held breast pump, a disc-shapedsupport member 56 is provided to support one or more sheets of absorbentor adsorbent material, such as a nitrocellulose membrane 39, in closeproximity, or in contact with, the nipple 16 when the device is in useand suction is applied to the breast. As shown in FIGS. 16 and 18, thesheet is preferably secured by an upper retainer ring 66 fixedly orremovably seated against an opposing surface (exemplified by acircumferential retainer groove 270) with the margin of the sheetsandwiched therebetween. This holds the sheet in place against negativepressure that may pass through the sheet when vacuum pressure is appliedthrough the engaging member and to secure the sheet in position when thenipple impinges against it. The retainer ring may be integrally joinedwith the support member which may comprise a disposable refill, or thering may be separable and the support member may comprise a reusablecassette for receiving replacement sheets. In an alternative design,there is no upper retaining ring and the membrane 39 or filter 40 simplyrests upon the support member 56 or is removably coupled directlytherewith (e.g., by wetting or gluing to create a temporary bond betweenthe sheet and upper support member surface, or by appropriately sizingthe sheet so that a peripheral edge of the sheet engages a surface ofthe support member in a friction or detent fit. Typically, the sheet isreadily removable from the support member for processing, e.g., by handor by using forceps or other conventional handling tools to disengagethe filter or membrane.

Typically, the support member 56 for use with the hand-held breast pumpdevice 10′ includes air channels 80 that pass through the body of thesupport member to allow vacuum pressure applied from the vacuum pump 160to reach the engaging member 14 of the pump during operation (i.e., bypassing from the pump through the sample collection housing 30 and airchannels to the engaging member). Likewise, the air channels allowventing of the engaging member for disengagement from the breast 17after use. In certain embodiments, a plurality of two-three or more airchannels are provided, which may be centrally located relative to adisc-shaped body of the support member as depicted in FIG. 18. The airchannels can serve a dual purpose as channels for passage or transfer offluids and/or fluid-suspended particles, including cells and cellularcomponents, between the breast engaging member and the sample collectionhousing 30.

In one related aspect of the invention, cellular materials fromexpressed breast fluid are first collected on a primary solid phasesample collection medium (e.g., a membrane 39 or filter 40) mounted infixed relation to the engaging member 14 (e.g., by a support member 56).In a secondary sample collection or processing step, the cellularmaterials are removed or flushed from the primary sample collectionmedium into a secondary solid phase sample collection medium (e.g., asolid phase sample collection template such as a plastic or glass slide,slip, tube, well or vial), optionally coupled with the support memberand/or sample collection housing. The cellular materials can be directlytransferred from the primary medium onto or into the secondary medium,for example by flushing the cellular materials from the primary medium(e.g., using physiological solutions, fixatives, etc.) directly into thesecondary medium (e.g., a recess, well, vial or other receptacle coupledwith or inserted into the support member or housing).

In one exemplary embodiment, cellular materials are collected fromexpressed breast fluid onto a primary sample collection mediumcomprising a filter, particulate medium, or nitrocellulose membrane 39.When a nitrocellulose membrane is used, the cellular materials arewashed after primary collection from the membrane by a flushing rinse.For example, cytology fluid or another desired rinse liquid is used totransfer the cells from the primary collection medium into a secondarycollection medium, typically a fluid-retaining well or reservoirintegrated within, or coupled with, the support member 56 or the samplecollection housing 30.

In more detailed embodiments of the invention, a nitrocellulose membrane39 is employed for primary sample collection. The membrane is typicallyfluidly connected with the hand-held pump 10′ by seating or mounting thefilter on or within a support member 56, as described above.Nitrocellulose membranes are particularly well suited for collection ofproteins, polynucleotides, or other soluble or suspended constituents ofbreast fluid. In preferred aspects, a nitrocellulose membrane isselected and employed within the device for collection of cytologicalspecimens, particularly cells and cellular constituents. As illustratedin FIGS. 16-18, the filter (optionally supported by a support member) ispositioned within the breast engaging member 14 near the base 22 of theengaging member proximate to the patient's nipple when the engagingmember is seated against the breast. Comparing FIGS. 16 and 17,activation of suction pressure by manual operation of the device drawsthe nipple 16 into closer proximity to, or into actual physical contactwith, the membrane, whereby the expressed fluid is efficientlytransferred to the membrane.

Due to the fragility and fine porosity of nitrocellose membranes 39, itmay be necessary to employ additional measures to protect the membraneagainst negative vacuum pressure and contact with the nipple 16 duringoperation of the pump 10′. In this regard, uniquely designed membranesand filters are provided for use within the device which featureperforations or slits that disrupt the planar surface of the membrane orfilter to facilitate air passage therethrough and impart structuralflexibility against mechanical perturbation. In one example, radialslits 280 emanate from a central disc portion 282 of the membrane orfilter (FIG. 24) to allow passage of air through the membrane or filterduring vacuum pressurization and to increase structural flexibility ofthe membrane or filter. Alternatively, one or more spiral perforations284 (FIG. 25) or transverse slits 286 (FIG. 26) may be cut or stamped inthe membrane or filter to achieve similar improvements in terms ofpermeability and flexibility.

Where the target constituent for collection from the expressed fluid iswhole cells or cellular components, these materials may be partitionedonto the surface of a membrane or filter, typically a nitrocellulosemembrane 39, for further processing and cytological examination. Forthis purpose, nitrocellulose membranes are employed which have asufficiently small pore size to retain the cells or cellular debris onthe outer (i.e., facing the nipple) membrane surface. For example,nitrocellulose membranes having a pore size of between about 0.5μ and5.0μ, preferably between about 1.0μ and 2.0μ, are useful to partitionwhole cells on their surfaces.

As noted above, a fluid-retaining recess, well or reservoir may befluidly connected to either the support member 56 or the samplecollection housing 30 of the handheld pump device 10′ for primary and/orsecondary sample collection. In certain embodiments, the fluid-retainingreservoir comprises an integral, defined compartment or enclosure withinthe sample collection housing for receipt of breast fluid and/orconstituent samples thereof, including cytology specimens that may bewashed into the reservoir after primary collection, as described above.Alternatively, the fluid well or reservoir can be a separable componentof the sample collection housing, e.g., in the form of a flexible lineror rigid fluid reservoir member of the housing removably connected witha complementary housing member that partially or completely encloses orotherwise engages the fluid reservoir member.

Thus, as depicted in FIGS. 16 and 18, certain embodiments of theinvention employ a removable fluid reservoir member 288 of the housing30 for secondary sample collection of breast fluid components, includingcytology specimens. In preferred embodiments, the removable reservoirmember is provided in the form of a rigid sample collection tube orvial, exemplified by a standard cytology vial (i.e., a container havingapproximately the same general shape and dimensions as a standardcytology vial). The tube or vial is removably connected with acomplementary housing member, for example an outer casing member 290 ofthe housing that partially or completely encloses the vial. Preferably,the tube or vial is sealably coupled with the outer casing member, forexample by partially or completely nesting the vial within the outercasing member to form an airtight coupling therewith.

In various specific embodiments, the tube or vial engages an inner wall292 of the casing member 290 and forms a generally airtight seal againstit. For example, the casing member and vial may be complementarily sizedand dimensioned to provide substantially airtight contact between theinner wall of the casing member and an outer wall 294, or a top end 296or bottom end 298, of the vial when the casing member and vial arecoupled to form the assembled housing. In certain embodiments, the outerwall of the vial features a circumferential ridge or fin 299 thatengages and thereby makes a circumferential airtight seal against theinner wall of the casing member when the vial is nested with the casingmember (see, e.g., FIGS. 16-20). In more detailed embodiments, the finor ridge is replaced by a flexible O-ring 300 that seats in acircumferential O-ring groove 302 on the outer surface of the vial andforms a circumferential seal with the inner casing wall.

The purpose of the sealable coupling between the outer casing 290 andremovable fluid reservoir 288 members of the housing 30 is to directvacuum pressure from the vacuum pump 160 to the breast engaging member14 in a path that includes the fluid reservoir member of the housing 30.In this manner, the removable reservoir is both gaseously and fluidlyconnected with the engaging member to facilitate secondary samplecollection. To accomplish these objectives, the removable reservoirmember is modified to include one or more air ports 310 that form agaseous connection between the lumen of the reservoir (which is in turnconnected to the engaging member, optionally via air channels 80 througha support member 56) and the vacuum pump. This allows the reservoirmember to remain in place during primary and/or secondary samplecollection, to function as both a conduit for vacuum pressuretransmission to the breast and a receptacle for fluid sample materials(e.g., to directly collect expressed fluid or as a secondary collectionmedium to receive primarily collected sample materials washed orotherwise transferred from the primary sample collection medium).

In the latter context, the removable reservoir member 288 of the housing30 may communicate for both fluid and gaseous transmission directly withthe breast engaging member 14 of the device, or indirectly by way of theair channels 80 in the support member 56 optionally coupled with theengaging member. As noted above, the air channels can serve a dualpurpose as channels for vacuum pressure transmission as well as transferof fluids from the primary sample collection medium, through the airchannels, into the removable reservoir (as exemplified by transfer of“wash” fluid containing cells and cellular components flushed from aprimary collector, e.g., a nitrocellulose membrane, mounted in thesupport member, through the air channels, and into the secondarycollection fluid reservoir). To facilitate this and related purposes,the channels may extend through tubular basal columns 311 or other fluidconnection ports that extend from the support member toward, or into, alumen 314 of the reservoir member of the housing.

In the embodiment of a hand-held pump device 10′ illustrated in FIGS.16-20, the reservoir member 288 is a cytology vial removably, sealablycoupled with the outer casing member 290 to form the assembled housing30. To achieve this sealable coupling, the outer wall 294 of the vialfeatures a circumferential fin 299, or a flexible O-ring 300 seated in acircumferential O-ring groove 302, that forms a circumferential sealwith the inner wall 292 of the casing member (see, e.g., FIGS. 16, 19and 20). The vial incorporates one or more air ports 310 thatcommunicate between the outer wall and the inner lumen 314 of the vialto form a gaseous connection between the lumen of the vial, the vacuumpump 160, and the breast engaging member 14. Preferably, multiple airports are provided, which are located on the side of the vial positionedbelow (i.e., toward the bottom end 298 of the vial) the ridge or O-ringthat forms the gaseous seal with the wall of the casing member.Alternatively, the air ports can be located at other positions on thevial, e.g., in a lower side wall or floor wall 318, provided theposition is suitable to maintaining the desired path of vacuum pressureflow and retaining fluids within the reservoir. In this context, it isnoted that certain embodiments of the fluid reservoir member of thehousing will dictate changes in the position of the air port(s). Forexample, where a cytology vial-shaped reservoir is selected, as shown inFIGS. 16 and 20, the floor wall may be raised relative to the bottom end298 of the vial, to reduce the sample volume of the vial for handlingand processing purposes. To accommodate this and other designs, the airports are desirably positioned in the outer (side) wall 294 of thereservoir between the floor wall and the sealing flange 299 or O-ring300.

Referring to FIGS. 19 and 20, further modifications of the removablefluid reservoir member 288 of the housing 30 provides for multi-purposeuse of the reservoir member for sample collection in a clinical setting,as well as sample storage, transport and/or processing in a laboratorysetting. To facilitate these latter purposes, the removable reservoirmember may be provided with closure means for closure of the reservoirafter sample collection is completed—to prevent sample contamination andspillage. Taking the cytology vial reservoir for example, the top end296 of the vial may be adapted to provide a sealable primary closure forthe main opening of the vial. For example, the vial top end can beprovided with complementary threads 320 or other closure means toreceive a conventional cap that is sized and dimensioned (e.g.,complementarily threaded) to sealably engage the vial top end. Inconjunction with this use, and further considering the novel adaptationof the fluid reservoir member to provide a secondary opening (i.e., theintegral air port 310 that functions as a vacuum connection), thereservoir is also equipped with secondary closure means to sealablyclose the air ports after sample collection. A variety of air portconfigurations are contemplated, which can be sealed using acommensurate variety of closure mechanisms. For example, the air port orports can be sealed using a flexible (e.g., rubber) stopper shaped anddimensioned to sealably plug into the port opening. Alternatively, theport can be closed by an adhesive seal or sticker that adheres to theouter wall 294 of the reservoir member surrounding the port opening.

With respect to the latter secondary closure design, the inventionprovides a combined closure and labeling device 324 which functions bothas a secondary closure mechanism to seal the air port 310 of theremovable reservoir and as a labeling template to provide a convenientwriting surface for sample labeling (see, e.g., FIGS. 21 and 22). Thisaspect of the invention may be achieved, for example, by providing anyof a variety of adhesive closure/labeling tabs that may be directlyapplied to sealed air port after sample collection. This simple type ofclosure/labeling tab can, for example, be provided as a separateadhesive sticker having a first, closure-forming surface 325 bearing anadhesive coating 326 on at least a portion of said surface, forapplication over the air port to form a seal, e.g., by adhesive contactwith the outer wall 294 of the removable reservoir. The adhesive coatingpreferably forms a seal that is resistant to disruption by contact withaqueous solutions and other materials present in the collected samples,for example buffers and fixatives. The adhesive coating is alsopreferably shielded before use by a protective tab or other protectivesurface 327 that covers the coating and is removed therefrom prior touse to expose the adhesive coating.

The closure/labeling device 324 has a second, labeling surface 328opposite the closure-forming surface 325 that is made of a blanktemplate material suitable for receiving a stable, ink or graphiteimprint thereon. For example, the second surface may be made ofcellulose or other fibrous material adapted for imprinting sample dataupon the surface thereof using a pen, pencil or other writing implement.Alternatively, the second, labeling surface may be made of plastic orother material adapted for retaining data imprinted in permanent ink(e.g., using an alcohol-based marker). When the adhesive tab is appliedover the air port, the blank template material of the second, labelingsurface of the tab is facing outward and at least a portion of thesurface covers a smooth portion of the outer wall of the removablereservoir 288, to allow the doctor or technician to imprint a clear datarecord on the labeling surface.

In more detailed embodiments, the closure/labeling device 324 is affixedto the removable reservoir 288 during operation of the hand-held breastpump 10′ in a first, open configuration that leaves the air port 310uncovered for transmission of vacuum pressure (see, e.g., FIG. 21). Theclosure/labeling device that is thus pre-attached to the reservoir canbe manually repositioned, or otherwise manipulated, after samplecollection to a second, closed configuration to form a seal or closureagainst the air port (see, e.g, FIGS. 21-23). In the exemplaryembodiment shown in FIGS. 19-23, the closure/labeling device comprisesan adhesive strip that is folded in a first, open configuration (FIG.21) to form two layers-an inner layer 330 that is affixed to thereservoir proximate to, but not covering, the air port, and an outerlayer 332 that folds back over the inner layer in the openconfiguration. In this embodiment, the outer layer of the strip providesboth the first, closure-forming surface 325 with the adhesive coating326 for securing closure of the strip, and the second, labeling surface328 formed of the blank template material opposite the closure-formingsurface. In the open configuration, the outer layer is optionallysecured in the folded-back position against the inner layer byengagement of the labeling surface with the inner layer, for example bya second adhesive coating 336 on the inner layer that holds the twolayers together in the folded position. Also optionally, the adhesivecoating 326 of the first, closure-forming surface may be protected inthe open configuration by folding an end segment 338 of the outer layer325 bearing the adhesive coating 326 for closure back on itself, so thatthe closure forming surface 325 provides the protective surface 327 toshield the adhesive prior to closure (as shown in FIGS. 21 and 22). Tomanipulate this closure/labeling device into the second, closedconfiguration, the end segment can then be lifted and pulled outward torelease the outer layer 332 from the inner layer 330 and to unfold theend segment to separate the adhesive coating 326 on the closure-formingsurface 325 from the protective surface 327. The outer layer 332 is thenunfolded away from the inner layer and wrapped around the reservoir sothat the closure-forming surface covers the air port to form a closurethat is water-tight or water-resistant to effectively prevent samplespillage from the reservoir and contamination. In this context, closureis effectuated by direct apposition of the adhesive coating 326 to theouter wall 294 of the reservoir surrounding the air port, or by drawingthe outer layer tight across the air port and annealing the adhesivesurface back upon the inner layer. In either case, manipulation of theclosure/labeling strip to the closed configuration positions thelabeling surface 328 to face outward for easy recordation of sampledata. In yet additional detailed embodiments, the closure/labeling stripcan be better secured against dislodgement during loading and removal ofthe reservoir 288 by including one or more circumferential guide ridges340 (see FIGS. 19 and 20) on the exterior of the reservoir, to shieldthe closure/labeling strip from mechanical dislodgement and/or toincrease the fidelity of the closure against leakage.

In related aspects of the invention, a novel breast fluid collectionreservoir is provided for use within a mammary fluid collection deviceof the invention, which incorporates the foregoing features of theremovable reservoir member of the sample collection housing. The novelcollection reservoir as described in the foregoing passages is usefulwithin the breast fluid collection methods of the invention, as well aswithin various sample handling, processing, and diagnostic assay methodsperformed in the laboratory subsequent to collection of a breast fluidsample.

The novel breast fluid collection reservoir of the invention istypically provided in the form of a rigid tube or vial, for example inthe form of a modified cytology vial, having a top end which defines aprimary opening for access to the sample. The reservoir furthercomprises an outer reservoir wall, typically a cylindrical sidewall of atubular reservoir closed at a bottom end thereof, e.g., by a floor wall,as described above. The outer reservoir wall defines one or more airports that communicate between the outer wall and an inner lumen of thevial. In more detailed embodiments, the fluid-retaining reservoircomprises a removable fluid reservoir member of a sample collectionhousing of a mammary fluid collection device. Typically, reservoirmember is a rigid sample collection tube or vial removably connectedwith an outer casing member of the sample collection housing of thecollection device.

Within this aspect of the invention, the reservoir may be adapted forremovable, sealable connection with the outer casing member of saidhousing, to form an airtight coupling therewith. In certain embodiments,the fluid-retaining reservoir is a cytology vial sealably connectablewith the outer casing member to form the airtight coupling. For example,the fluid-retaining reservoir can be removably nested within the casingmember to form a substantially airtight contact between an inner wall ofthe casing member and an outer wall, or a top or bottom end, of thereservoir member. To achieve this function, the outer wall of thefluid-retaining reservoir may be provided with a circumferential ridge,fin or O-ring adapted to engage and make a circumferential airtight sealagainst the inner wall of the casing member.

In more detailed aspects, the fluid-containing reservoir member for usewithin the devices and methods of the invention includes a closuredevice for closing the reservoir after the sample of mammary fluid isintroduced therein, to prevent sample contamination and spillage. Theclosure may comprise a simple cap adapted to sealably engage a top endof the reservoir. The cap or similar “primary closure” may extend tocover the air port(s) of the reservoir, which may be contiguous with thetop opening of the reservoir or separate therefrom. In alternativeembodiments, secondary closure means may be provided which arespecifically adapted for closure of the air port(s). The secondaryclosure means may comprise a plug, cap or adhesive seal or sticker sizedand constructed to engage or adhere to the outer wall of the reservoirmember at or surrounding the air port opening(s) to form the closure.

In certain embodiments, the secondary closure means comprises a combinedclosure and labeling device which functions as a secondary closuremechanism to seal the air port(s) of the reservoir and as a labelingtemplate to provide a writing surface for sample labeling. Often, thesecondary closure means comprise a combined closure and labeling tab orsticker for application to the outer wall of the reservoir to seal theair port after the sample is collected. The tab or sticker has a first,closure-forming surface for application over the air port to form a sealby juxtaposition or adhesive contact with the outer wall of thereservoir, and a second, labeling surface opposite the closure-formingsurface made of a blank template material for imprinting writteninformation thereon. The first, closure-forming surface typically has awater-insoluble adhesive coating on at least a portion of the surface.In more detailed embodiments, the tab or sticker is pre-attached to theremovable reservoir member in a first, open configuration and can bemanually repositioned or otherwise manipulated after sample collectionto a second, closed configuration to form a seal or closure against theair port(s), as described in detail above.

In yet additional, related aspects of the invention, methods for breastfluid sample collection, sample handling, and/or sample processing areprovided which incorporate the novel fluid-retaining reservoir adaptedfor use with a hand-held breast pump 10′ of the invention. These methodsinclude, generally methods for collecting breast fluid samples whichinvolve collecting expressed mammary fluid in a modified fluid reservoiras described above. Additional methods involve loading and removal of amodified fluid-retaining reservoir within a hand-held breast pump,according to the above description. Related methods include anadditional step of securing the primary and/or secondary closure meansof the reservoir after a sample of mammary fluid, or a componentthereof, is collected therein.

Related to these methods, the invention provides additional methods forhandling or processing biological samples of mammary fluid, orcomponents thereof, for use in a diagnostic assay to detect or quantifya breast disease marker in the sample. The methods generally involveproviding or obtaining the biological sample of mammary fluid or amammary fluid component in a specialized fluid-retaining reservoiraccording to the above description. The sample may be initiallycollected by oxytocin induction or by application of a breast pump 10,10′ of the invention without oxytocin priming. The reservoir istypically provided as a flask, vial, or tube that has a top end defininga primary opening for collection of, and later access to, the mammaryfluid sample, and an outer reservoir wall that defines one or more airports communicating between the outer wall and an inner lumen of thevial.

In more detailed handling and processing methods, the reservoirincorporates specialized closure means, for example a cap that securesthe top end of the reservoir and secondary closure means to sealablyclose one or more air port(s) of the reservoir, to close the reservoirafter the sample is collected and thereby prevent sample contaminationand spillage. Typically, the fluid-retaining reservoir is a modifiedcytology vial adapted as a removable reservoir member integrated with ahand-held mammary fluid collection pump 10′ as described above. Moredetailed handling/processing methods employing the novel reservoirinclude the step of accessing said sample within the reservoir totransfer or process the sample for detection or quantification of abreast disease marker. Additional methods include one or more steps ofprocessing the sample to detect or quantify the breast disease marker.

In additional detailed embodiments, the step of processing the samplefor marker detection comprises fixing or staining cells or cellfragments in the sample, before or after transfer of the sample from thereservoir, e.g., for cytological analysis. Yet additional methodsinvolve exposing the sample in the reservoir or after transfer to aprocessing reagent, e.g., a fixative, labeling reagent, buffer, etc., toprepare sample components, including whole mammary fluid, whole cells,cell fragments, cell membranes, purified proteins, bulk proteins,glycoproteins, peptides and/or polynucleotide components, for furtherprocessing, which may include detection or quantification as theselected breast disease marker(s).

As noted above, certain modular designs for the hand-held breast pump10′ of the invention feature a separate breast engaging member 14provided as a funnel shaped component adapted for removable coupling toone or more interconnecting components of the device for cleaning andinterchanging of parts. Typically, the breast engaging member isremovably coupled with the sample collection housing 30. In oneembodiment shown in FIGS. 16-18, the housing is comprised of multiplemembers, exemplified by an outer casing member 290 and a removable,fluid reservoir member 288. As illustrated in FIG. 18, the engagingmember may be directly coupled to the fluid reservoir member, which isin turn engaged by a sealable connection (e.g., a sealable compressionfit) with the outer casing member of the housing as described above. Inthis context, one alternative coupling design is for the engaging memberto be fitted with mounting threads 340 or other coupling means to couplewith complementary threads 320 or other closure means on the top end 296of the removable reservoir (e.g., cytology vial). In this manner, theclosure means of the removable reservoir, adapted to receive aconventional cap that sealably engages the reservoir top end, serves thedual purpose of coupling the engaging member 14 with the remainingmodular components of the device. Alternative coupling means are ofcourse contemplated as well, as exemplified by a simple pressure fitcoupling to removably couple mated ends of the engaging member andreservoir member. To facilitate stable coupling of the engaging memberwith the sample collection housing 30, the housing may be furtherelaborated to include flared extensions 350 terminating in outwardlyreflected feet 352 collectively shaped and dimensioned to engage aninner, circumferential groove 354 underlying the flared or reflectedterminal edge 18 of the engaging member (see, e.g., FIG. 18).

In addition to the foregoing features, the hand-held breast pump 10′ ofthe invention may optionally include any of the alternative featuresdescribed above for the general-purpose breast pump 10, includingdifferent solid phase sample collection media and support member 56designs. Thus, the support member for use with the hand-held pump canalso include a fluid-retaining well which may be optionally filled witha desired solution, such as a buffer, a solution containing a probe,cross-linking agent, blocking agent, denaturing agent, etc., tofacilitate sample collection, handling, and/or processing. Alternativedesigns and configurations of the housing 30 and/or support member 56are also provided which vary with the type of solid phase samplecollection medium used. For example, when a particulate solid phasesample collection medium 41 (e.g. beads, resins, or microspheres) isused, the medium may be enclosed in a cartridge 82 removably mounted to,or integrated within, the support member or otherwise removablyconnected to the sample collection housing 30, as described in detailabove. It is also contemplated to adjustably mount the solid phasemedium relative to the housing 30 of the hand held pump device, so thatthe collection medium can be moved closer to, or farther away from, thebase 22 of the engaging portion 14 of the pump 10. In this regard,various designs are contemplated commensurate with the above descriptionto provide a reciprocating mechanism which adjustably moves the solidphase collection medium in closer, or more distant, proximity to thenipple when the hand-held breast pump is engaged therewith.

Within more detailed aspects of the invention, the hand-held breast pump10′ typically incorporates a compact vacuum pump housing 410 whichstructurally and functionally integrates the vacuum pump 160 with thesample collection housing 30 and, in turn, with the engaging member 14(see, e.g., FIGS. 16-18). The vacuum pump housing is in turn coupledwith, or is modified to include, a vacuum pump actuating mechanism. Theactuating mechanism may be in the form of a switch, button, lever orother actuation device suitable for use with the selected vacuum pump.As noted above, a variety of vacuum pumps may be incorporated within thebreast pump device, including any manual or electric, piston, hydraulicor diaphragm pump of suitable size and dimension for incorporation inthe handheld pump vacuum housing. In the exemplary embodiment depictedin FIGS. 16-18, the vacuum pump is a conventional diaphragm pump, andthe pump actuation mechanism is a simple hand lever 420 pivotallyconnected to the pump housing or other suitable connection point.

Exemplifying this aspect of the invention, the pump housing isoptionally coupled with, or extended to include, an opposing handle 422to facilitate depression of the hand lever by gripping and manualclosure of the lever toward the handle (compare FIGS. 16 and 17). Thehandle is preferably molded or cast as an integral extension of thevacuum pump housing. In more detailed embodiments, exemplified in FIGS.16-18, the vacuum pump housing and handle are molded or cast together asan integral unit or modular component with the outer casing member 290of the sample collection housing 30. The handle defines a pivot recess430 or detent that pivotally receives a pivot head 432 or shaft joinedor integrated with the pump actuation lever (as shown in FIGS. 16 and17).

In this manner the pump actuating lever 420 is pivotally connected tothe pump housing 410 or handle 422 by a pivotal connection 430, 432 thatjoins the actuation lever and pump housing in an easily assembledfashion, e.g., by snap fitting the pivot head 432 of the lever into thepivot recess 432 of the handle. The actuation lever easily andeffectively actuates the vacuum pump 160 by depressing the lever to drawa flexible diaphragm member 440 downward, away from a primary vacuumchamber 442 connected with, or integrated within, the sample collectionhousing. In the embodiment shown in FIGS. 16-18, the primary vacuumchamber is integrally formed as a channel within the vacuum pump housingproximate the flexible diaphragm member and extending to a communicatingport 444 opening to the inner wall 292 of the outer casing member 290 ofthe housing. The remainder of the vacuum path (i.e., through the outercasing member to the air port 310 of the removable fluid reservoirmember 288 of the housing into the lumen 314 of the reservoir,optionally through air channels 80 of the support member 56, and to thebreast engaging member) is described above.

To engage and move the flexible diaphragm member 440 in this fashion,the actuation lever engages a diaphragm retraction mechanism, forexample comprising a reciprocating shaft 450 or piston sealablyconnected through the diaphragm to a diaphragm-engaging head 452, tocollectively translate depression movement of the lever to downwardretraction of the flexible diaphragm member (compare, e.g., FIGS. 16 and17). These components of a diaphragm pump are conveniently housed withina protective pump cover 460 which engages a complementary rim 462 orother coupling surface of the pump housing 410 by a complementaryengagement fitting (arrows 464). This coupling may optionally serve tosealably anchor the flexible diaphragm member, e.g., by sandwiching aperipheral edge 466 of the diaphragm between the cap and housing asshown in the figures. Optional pump devices, actuation mechanisms, andpump housing designs are contemplated within the invention, which arewithin the level of skill in the art to engineer for use with thehandheld breast pump devices disclosed herein.

As noted above, mammary fluid expression and collection using thedevices of the invention may be facilitated in certain instances byprior or concurrent administration of the peptide hormone oxytocin, oran analog thereof, in an amount that is effective to stimulatemyoeptithelial contraction in the alveolar gland ducts of the breast tofacilitate expression of the mammary fluid from the nipple. Preferablythe oxytocin preparation is administered intranasally and isadministered in an amount that is intranasally effective to stimulateexpression of mammary fluid from the nipple. Alternatively, anintramuscular or intravascular injection of oxytocin can effect the samemyoepithelial contraction response as the intranasal administrationroute. The amount, timing and/or mode of oxytocin administration may beadjusted on an individual basis depending on such factors as menstrualcycle stage, use of birth control or hormone replacement therapy,pregnancy history, age of onset of menarche, ethnicity and other factorsknown to affect an individual's propensity for breast fluid expression.

Oxytocin is a peptide hormone of pituitary origin that is naturallyreleased into the bloodstream of lactating women in response tosuckling, and stimulates contraction of myoepithelial cells in themammary alveoli and ducts to cause milk ejection (Cobo, J. Perinat. Med.21:77-85, 1993). The drug has also been widely used for stimulatinglabor in pregnant women, due to its activity of stimulating uterinecontractions (Satinet al., Am. J. Obstet. Gynecol. 166:1260-1261, 1992).For these reasons, the pharmacology of oxytocin has been thoroughlyinvestigated, including detailed studies of effective dosages, half-lifeand potential side effects.

For use in the present invention, an oxytocin preparation is providedfor intranasal, intramuscular, or intravenous administration thatcontains oxytocin in a biologically suitable, liquid carrier. As usedherein, “oxytocin” refers to natural or synthetic oxytocin andbiologically active derivatives and analogs thereof. Naturally occurringoxytocin from mammalian sources is of course suitable, as are otherknown, naturally occurring oxytocin-like peptide analogues and theirsynthetic counterparts having similar activities for stimulatingalveolar-ductal myoepithelial contraction. Preferably, the oxytocin usedwithin the invention is a simple peptide hormone comprising a cyclicpeptide, the peptide having a well defined ring portion(Cys-Tyr-Ile-Gln-Asn-Cys) and tail portion (Pro-Leu-Gly). However,numerous derivatives and analogues are known, or readily obtainable, inthe art, e.g., derivatives or analogues having amino acid truncations,deletions or substitutions at one or more residues of the peptide andwhich exhibit substantially the same activity as naturally occurringoxytocin (i.e., having at least 75%, and preferably 85%-95% or more,activity compared to that of native oxytocin for stimulatingalveolar-ductal myoepithelial contraction). The most economic oxytocinpreparations for use within the invention contain a synthetic oxytocin(e.g. Pitocin® or Syntocinon® available from various providers, forexample Sandoz (Basel, Switzerland) and United States Pharmacopeia.Alternate benefits may be obtained with the use of a long-actingoxytocin analog within the methods of the invention. The utility andpharmacokinetics of such analogs, exemplified by the peptide analogcarbetocin, are described in detail in U.S. patent application Ser. No.09/481,058 filed Jan. 11, 2000 (incorporated herein by reference).

For use with the methods, devices and kits of the invention, a preferredoxytocin preparation contains approximately 40 USP units of oxytocin perml of liquid carrier. Preferred liquid carriers are biologicallycompatible solutions, such as a lactated Ringer's solution or otherphysiologically balanced, sterile, non-toxic and non-irritativesolution. To administer the oxytocin intranasally, a standard nasalsqueeze bottle is used, which delivers approximately 0.5 ml of theoxytocin preparation into the patient's nostril when squeezed. Theoxytocin is absorbed by the nasal mucosa into the systemic circulationwhere it reaches and acts specifically on the myoepithelial cellssurrounding the alveoli of the breast and making up the walls of thelactiferous ducts, causing their smooth muscle fibers to contract andforce any fluids present into the large ducts or sinuses where it can beexpressed from the nipple spontaneously onto a sample collector or bythe further action of a breast pump. Intranasal application of the spraypreparation is therefore a practical and effective method ofadministration. The half-life of oxytocin in the human bloodstream isextremely short, estimated to be about 10-15 minutes or less, due to itsrapid removal from plasma by the kidney, liver, and mammary gland, andthe time to pharmacokinetic and clinical steady state is readilydetermined depending on the mode of administration (e.g. bolus dosage,repeat administration, or steady infusion). (See for example, Gonser,Arch. Gynecol. Obstet. 256:63-66, 1995; and Orhue, Obstet. Gynecol.83:229-233, 1994, each incorporated herein by reference in itsentirety). It is therefore a routine matter to determine an appropriateconcentration and dose of the oxytocin preparation to administer aneffective amount (either intranasally effective, intravenouslyeffective, or intramuscularly effective) of the oxytocin to causeexpression of mammary fluid with or without the assistance of a breastpump. (See for example, Newton, Ann. N.Y. Acad. Sci. 652:481-483; Mena,Neuroendocrinology 61:722-730, 1995; Gonser, Arch. Gynecol. Obstet.256:63-66, 1995; Orhue, Obstet. Gynecol. 83:229-233, 1994; Satin et al.,Am. J. Obstet. Gynecol. 166:1260-1261, 1992; and Satin et al., Obstet.Gynecol. 83:234-238, 1994, each incorporated herein by reference in itsentirety).

Although not all female patients are expected to be responsive tointranasal oxytocin stimulation, an intranasally effective amount ofoxytocin for the purposes of the invention can be readily determined. Asused herein, an intranasally effective amount of oxytocin is an amountof oxytocin sufficient to intranasally stimulate the expression of atleast 3 μl of mammary fluid in at least 50% of non-lactating femalepatients with the aid of negative pressure to the nipple of between50-200 mm Hg applied by the breast pump (up to 45 min after a firstadministration of the oxytocin spray). It may be necessary, and indeedpreferred, to administer a low, preliminary dose of oxytocin to thepatient, for example a single spray of a 40 Unit/ml oxytocin solution ineach nostril, or multiple sprays of a lower concentration oxytocinpreparation, and thereafter wait to determine a particular patient'ssensitivity. If there is no reaction with an initial application of thebreast pump after a short post-administration period of 2-15 minutes,and preferably 2-5 minutes, a booster dose of the oxytocin spray may beadministered and the pump reapplied. In this way, the clinician canmodulate the dosage to each patient's varying sensitivity, and therebyminimize potential adverse side effects. Alternatively, an effectivedose of intramuscular or intravenous oxytocin can be used according tothe same dosage determination and administration principles in patientswhere intranasal administration fails or is otherwise contra-indicatedas a preferred mode of administration.

As noted above, the amount, timing and/or mode of oxytocinadministration may be adjusted based on specific factors known to renderindividuals more or less sensitive to induction of breast fluidexpression. These factors are generally well known in the art, andinclude, for example, menstrual cycle stage, use of birth control orhormone replacement therapy, pregnancy history, age of onset ofmenarche, and ethnicity, among other factors.

Thus, in one aspect of the invention, methods for obtaining a biologicalsample from a patient and/or determining the amount of a breast diseasemarker in a biological sample from breast fluid are provided whichinclude a step of determining a menstrual stage of the patient. Based onthe determined menstrual stage, a drug administration protocol isselected having a predetermined oxytocin dosage, timing and/or frequencyof oxytocin delivery, and/or mode of oxytocin administration.

According to these methods, one or more variables of oxytocin dosage,timing and/or frequency of oxytocin delivery, and/or mode of oxytocinadministration are selected depending on whether the patient is stagedwithin one of five approximate menstrual phases. These phases include 1)a proliferative phase (characterized by a tight configuration of thealveolar lumena); 2) a follicular phase (characterized by a definedconfiguration of the alveolar lumena); 3) a luteal phase (characterizedby an open configuration of the alveolar lumena, with some secretion bythe alveolar cells); 4) a secretory phase (characterized by an openconfiguration of the alveolar lumena, with secretion by the alveolarcells); and 5) a menstrual phase (characterized by a distendedconfiguration of the alveolar lumena, with secretion by the alveolarcells).

It is generally not desired to conduct the methods of the invention forpatients staged in the proliferative or follicular stage of theirmenstrual cycle (approximately 3-7 days and 8-14 days, respectively).However, in some circumstances sample collection can be performed forthese individuals using high and/or repetitive doses of oxytocin andotherwise optimizing the breast fluid expression response by selecting aparticular mode of oxytocin administration, or combination thereof(e.g., intravenous administration followed by intranasaladministration). For patients staged in the luteal or secretory stage oftheir menstrual cycle (approximately 15-20 days and 21-27 days,respectively), intermediate dosages of oxytocin are selected andrepetitive administrations are reduced or eliminated. For patientsstaged in the menstrual phase, dosages of oxytocin and repetitiveadministrations are reduced even further while still providing aneffective administration protocol to yield sufficient breast fluidexpression.

Determination of effective administration protocols for patients ofdifferent menstrual stages can also be readily achieved within theinvention. As used herein, an effective administration protocol yieldsat least 3 μl of expressed mammary fluid in at least 50% ofnon-lactating female patients at an equivalent menstrual stage with theaid of negative pressure to the nipple of between 50-200 mm Hg appliedby a breast pump up to 45 min after a first administration of theoxytocin spray. Various combinations of oxytocin dosage, timing and/orfrequency of oxytocin delivery, and/or mode of oxytocin administrationare contemplated, as can be readily determined by the skilled artisan inaccordance with the teachings herein. Likewise, it will often bepreferred to administer a low, preliminary dose of oxytocin to thepatient and thereafter wait to determine a particular patient'ssensitivity, even when an individual's menstrual stage has beendetermined and a particular administration protocol selected. Thus, ifthere is no reaction with an initial application of the breast pumpafter a short post-administration period, a booster dose of the oxytocinmay be administered and the pump reapplied. In this way also, theclinician can apply a first, stage specific dose of oxytocin andthereafter modulate the dosage, period of time between boosteradministrations, and/or mode of administration, to each patient'svarying sensitivity.

In other, related aspects of the invention, methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid areprovided which include a step of determining a non-menstrual stagepatient sensitivity index. Examples of such indices include 1) patientuse of hormone based birth control; 2) patient use of hormonereplacement therapy; 3) patient pregnancy history; 4) patient age ofonset of menarche; and 5) patient ethnicity. Other indices associatedwith sensitivity to induction of breast fluid expression are alsocontemplated. These factors can be determined by such routine steps aspatient consultation, evaluation of patient records, and clinical orlaboratory-based analysis (e.g., physical screening, measurement ofsex-steroid hormone levels, etc.) Based on a determined non-menstrualstage sensitivity index, an effective drug administration protocol isselected having a predetermined oxytocin dosage, timing and/or frequencyof oxytocin delivery, and/or mode of oxytocin administration, inaccordance with the methods described above. In yet additional methodsan effective drug administration protocol is selected by firstdetermining both a patient's menstrual stage and at least onenon-menstrual stage sensitivity index specific to the patient, andthereafter selecting an effective oxytocin administration protocol basedon these combined indices.

In yet additional methods within the invention, it may be preferred toconduct the foregoing sample collection methods in conjunction with aconventional mammographic procedure. In this manner, costs, time andpatient discomfort can be minimized. Further, by conducting the samplecollection immediately following a mammogram it is expected that breastfluid expression may be facilitated by breast manipulation during theinitial procedure. Additional steps to facilitate breast fluidexpression include manual breast massage and application of heat packsto the breast.

For mammary fluid collection using a breast pump 10, 10′ of theinvention, alone or in conjunction with oxytocin stimulation, the breastpump is applied and negative pressure is generated on the breast tofacilitate the expression of mammary fluid. Within the methods of theinvention, negative pressures of 50-200 mm Hg are preferred, and thesepressures are maintained, preferably intermittently, for approximately1-15 minutes, depending on the sensitivity of individual patients,oxytocin dosage and other factors. The volume of expressed mammary fluidwill vary depending on a variety of factors, including the time andpressure of breast pump administration, and other factors. For the leastsensitive breast marker assays of the invention, a volume of expressedmammary fluid of 300-500 μl is preferred to provide ample material forconducting the assay, and these volumes will be obtainable from asubstantial proportion of women treated according to the above methods.To express 300-500 μl of mammary fluid, some women will require repeatedstimulation treatments, perhaps requiring pooling of mammary fluidsamples obtained during multiple patient visits. However, for moresensitive assays of the invention, e.g. solid phase immunoassays, muchsmaller samples of 3 μl or less will be suitable to carry out theassays, particularly in the case of breast cancer markers that arenaturally secreted into the mammary fluid and are therefore expected tobe present in very high concentrations compared to, for example, breastepithelial cell surface antigens or intracellular antigens that are notsecreted.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1 Induction of Mammary Fluid Expression by Application of aNovel Breast Pump/Mammary Fluid Sample Collection Device

Within the present example, a hand-held breast pump device 10′ isemployed to collect a primary sample of mammary fluid componentscomprising whole cells and cell fragments for cytological examination.The doctor, technician or patient collects the breast fluid specimen bygrasping and operating the hand-held pump as described above tostimulate expression of the mammary fluid and collect a specimenthereof. This operation is a one-handed procedure, leaving the physicianor technician free to conduct additional activities with the other, freehand. In this regard, the compact hand-held pump design allows thedevice to be picked up and manipulated with one hand, to seat the breastengaging element against the breast, apply sufficient vacuum pressure tothe breast by manual operation of the vacuum pump to cause a suitablevolume of breast fluid to be expressed at or near the nipple, and tosimultaneously collect at least a primary sample of expressed breastfluid onto, or within, the solid phase sample collection medium (e.g., anitrocellulose filter) without additional manual steps or the need toremove the device from the breast or engage two hands in the operation.

In the present example, the hand-held pump device 10′ is employed tocollect a primary sample of a selected mammary fluid componentcomprising cells and other cellular materials, that are retained (e.g.,by retaining cells on a nitrocellulose membrane 39 or filter 40 coupledwith the breast engaging member 14 or sample collection housing 30) forfurther processing. The primary collected sample of cells and othermaterials is then transferred (e.g., by washing or manual transfer) to areservoir or other solid phase template, for further storage, processingand/or analysis. In more specific protocols, whole cells are separatedfrom expressed mammary fluid onto a nitrocellulose membrane 39 or afilter 40 secured in fluid connection with the breast engaging member 14a removable support member 56 mounted to the engaging member or samplecollection housing 30.

The cells are subsequently transferred or washed in fluid (e.g.,cytology fluid) to a second solid phase sample collection medium (e.g.,a slide, well, tube or vial), which may also be connected to, orintegrated with, the breast engaging member 14 or sample collectionhousing 30 as described above. In one protocol, the breast engagingmember is removed from the breast and a jet of cytological fluid isdirected against the filter on which the cells are retained. Thisflushes the cells off of the filter into an awaiting reservoir,typically a removable fluid-retaining reservoir. In this regard,uniquely designed membranes and filters are provided for use within thisprotocol which feature perforations or slits that disrupt the planarsurface of the membrane or filter to facilitate air passagetherethrough, to impart structural flexibility against mechanicalperturbation, and to allow cells flushed from a first side (facing thebreast) of the filter to a second side (facing internally, e.g., towardthe sample collection housing, when the filter is mounted with thedevice) for secondary sample collection. For example, radial slits 280,spiral perforations 284 or transverse slits can be made in the filter toallow passage of air and for cells partitioned onto the first surface ofthe membrane to be flushed through the membrane into the removable,fluid-retaining reservoir 288 seated in the sample collection housing30, for storage, shipment, and/or further processing directed towardcytological examination of the secondary collected sample.

EXAMPLE 2 Cytology in Biological Samples From Mammary Fluid

This example describes the use of conventional cytological techniques toidentify and classify breast diseases from samples obtained as describedin Example 1. Following collection of the sample, e.g. in afluid-retaining reservoir member, the sample may be further processed(e.g., by centrifugation wherein the reservoir member is in the form ofa modified cytology vial). Processed samples are then transferred to thecentral region of a clean glass microscopic slide, and a cover slip isslid over the sample to spread it along the surface of the slide. Theslide is allowed to air dry and then is fixed, for example in absolutealcohol, and stained with standard cytological stains, such as methyleneblue, hematoxyln and eosin, and other suitable stains.

The slides are then examined by light microscopy for evidence ofatypical growth of cells and clumps of cells, using well known methods,including those described in Diagnosis of Non-Palpable Breast Lesions:Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic andPrognostic Implications of Cytology by Jacqueline Mouriquand, S. KargerPub., July 1993; Breast: Guides to Clinical Aspiration Biopsy by TildeS. and Irwin K. Kline, Igaku-Shoin Medical Pub., May 1988; Cytopathologyof the Breast (Asop Theory and Practice of Cytopathology 5 by ShahlaMasood, American Society of Clinical Pathology, November 1995; FineNeedle Aspiration Cytology and Its Clinical Applications: Breast andLung by Philip S. Feldman, American Society of Clinical Pathology,November 1984; each incorporated herein by reference in its entirety.

EXAMPLE 3 Stimulation of Mammary Fluid Expression for Sample Collectionby Coordinate Administration of Intranasal Oxytocin in Conjunction withApplication of a Novel Breast Pump/Mammary Fluid Sample CollectionDevice

The foregoing sample collection protocol in Example 1, as well as othersample collection methods within the invention, may be practiced solelyby the use of a novel breast pump 10, 10′ of the invention.Alternatively, these sample collection procedures may be practiced inconjunction with the use of oxytocin or oxytocin analogs to facilitateor increase mammary fluid expression induced by operation of the breastpump. As incorporated within the invention, these methods involveapplication of the breast pump 10, 10′ to the breast, optionally coupledwith oxytocin administration in amounts effective to facilitate mammaryfluid expression in the patient. After the sample is collected, abioassay is conducted on the sample to determine the presence and/oramount of a selected breast disease marker, preferably a breast cancermarker or panel of breast cancer markers, in the sample.

Oxytocin nasal solution acts specifically on the myoepithelial elementssurrounding the alveoli of the breast and making up the walls of thelactiferous ducts, causing their smooth muscle fibers to contract andthus force any fluids present into the large ducts or sinuses where itcan be expressed from the nipple by the further action of a breast pump.The nasal spray is promptly absorbed by the nasal mucosa to enter thesystemic circulation. Intranasal application of the spray preparation isa practical and effective method of administration. Half-life ofoxytocin in the human circulation is extremely short, approximately10-15 minutes, and oxytocin is then rapidly removed from plasma by thekidney, liver, and mammary gland.

Because of the known effects of oxytocin to cause uterine contractions,pregnant women should not be treated by the methods contained hereinunless the benefits of testing outweigh the risk of inducing prematurelabor.

The oxytocin nasal solution contains a concentration of natural orsynthetic oxytocin, or a functional analog thereof such as carbetocin,that is intranasally effective in a selected volume of administeredspray to stimulate expression of mammary fluid from a nipple of thepatient when a breast pump is applied to the nipple to assist mammaryfluid expression. In the present example, a preferred oxytocinpreparation containing approximately 40 USP units of oxytocin per ml oflactated Ringer's solution is administered into the nose with thesqueeze bottle held in the upright position while the patient is in asitting position. One or two sprays are administered into each nostrilfrom a standard nasal squeeze bottle, which delivers approximately 0.5ml of the oxytocin solution per spray in a fine mist when the bottle issqueezed. The number and volume of sprays administered, as well as theconcentration of oxytocin in the solution, can be adjusted according towell known pharmacokinetic principles (See for example, Newton, Ann.N.Y. Acad. Sci. 652:481-483; Mena, Neuroendocrinology 61:722-730, 1995;Gonser, Arch. Gynecol. Obstet.256:63-66, 1995; Orhue, Obstet. Gynecol.83:229-233, 1994; Satin et al., Am. J. Obstet. Gvnecol. 166:1260-1261,1992; and Satinet al., Obstet. Gynecol. 83:234-238, 1994, eachincorporated herein by reference in its entirety) to ensure that theamount of oxytocin administered to the patient corresponds to anintranasally effective amount to stimulate the expression of at least 3μl of mammary fluid in at least 50% of non-lactating female patientsupon activation of the breast pump. For example, adjustments may bedesired in the number of sprays delivered b the patient, and/or thetiming of spray delivery, so that the clinician can modulate the dosageto each patient's varying sensitivity, and thereby minimize potentialadverse side effects. In the present example, a preliminary dose of asingle spray of the 40 Unit/ml oxytocin solution is delivered into eachnostril of the patient, and the administering clinician waits for ashort post-administration period of 2-3 minutes. After this period thebreast pump is applied, and the clinician determines whether or not theamount of oxytocin delivered was sufficient to facilitate or increasebreast pump-induced expression of mammary fluid. If additional fluidexpression is desired at this stage a booster dose of 1 or 2 additionalsprays of the oxytocin solution can be administered in each nostril, andthe pump reapplied after a 5-10 minute post-booster administrationperiod.

After the intranasally effective dose of the oxytocin is administeredand the clinician has allowed a suitable post-administration period toelapse for the oxytocin to reach and stimulate the targetalveolar-ductal tissue, the breast pump is applied. Negative pressuresof 50-200 mm Hg are applied in the area of the nipple and aremaintained, intermittently or continuously, for approximately 1-15minutes, depending on the sensitivity of individual patients, oxytocindosage and other factors. Alternatively, oxytocin can be administered byintramuscular or intravascular routes by well known means (OxytocinInjection (synthetic), USP; Wyeth-Ayerst Laboratories) to effect thesame response as intranasal administration.

EXAMPLE 4 Verification of Sample Quantity, Origin and Quality

Using either of the above methods of Example 1 or Example 3, volumes ofat least 3 μl of expressed mammary fluid can be collected in asubstantial population of non-lactating female patients. During or afterthe mammary fluid expression step, a biological sample is collected fromthe expressed mammary fluid as described above. For example, anitrocellulose filter may be placed within the breast pump in line witha path of the expressed mammary fluid into the pump, so that theexpressed fluid contacts the filter. Upon contact of the primary sampleof expressed mammary fluid with the filter, cells, proteins and otherdesired components of the mammary fluid adhere to the filter to form afilter-bound or filter-F retained biological sample for subsequentanalysis. Other suitable biological samples, including whole mammaryfluid samples, cytological samples of whole cells, membranes or othercellular components, and samples containing proteins, glycoproteins,peptides, nucleotides and other constituents of the primary mammaryfluid sample can be collected with appropriate modifications of theabove procedures, according to well known principles and methods.

To ascertain that the sample of mammary fluid is of mammary origin andis not corrupted by likely contaminants, one or more constituents ofnormal mammary fluid are assayed for. In the present example, an enzymethat is ordinarily present in mammary fluid, lysozyme, is assayed in themammary fluid sample to help confirm that the sample is of mammaryorigin. Lysozyme (muramidase) is an enzyme which hydrolyzes beta1,4-glycosidic linkages in the mucopolysaccharide cell wall of a varietyof microorganisms, which activity can be readily detected and quantifiedusing a routine, inexpensive assay. In the present example, Lysozyme ismeasured in the primary mammary fluid sample using the QuantiplateLysozyme Test kit (Kallestad, Chasta, Minn.). The assay employs thereagents and procedures provided by the manufacturer and specified indetail in the manufacturer's instructions, with the exception that amammary fluid sample is substituted in place of serum, urine or tears.Analysis of these results establishes that the sample contains lysozyme,which is a normal component of human serum, urine, saliva, tears, nasalsecretions, vaginal secretions, seminal fluid, and mammary fluid.

More specific assays are used in place of the lysozyme assay, or tosupplement lysozyme assay results, particularly where clinical data forhuman patients are being gathered. Other mammary fluid markers forsample verification that are more specific than lysozyme can be readilyincorporated within the invention, based on published and generallyknown information. In one example, the presence of cathepsin D isassayed using the monoclonal antibodies and methods disclosed inVetvicka et al., Biochem. Mol. Biol. Int'l. 30:921-928, 1993,incorporated herein by reference in its entirety). In another example,one or more human mammary epithelial antigens (HME-Ags) corresponding toglycoprotein components of the human milk fat globulin (HMFG) proteinare detected in the primary mammary fluid sample, or in the biologicalsample that is used in the breast cancer marker assay, using specificantibody probes, as described by Rosneret al., Cancer Invest. 13:573582,1995; Ceriani et al., Proc. Natl. Acad. Sci. USA 74:582-586, 1982;Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990, eachincorporated herein by reference in its entirety). In many cases, thesample verification assay can be incorporated within the breast cancermarker assay in a single procedure, for example as described below inExample 4, an assay for HME-Ags (wherein the HME-Ag findings areindicative of sample origin/quality, and also of the presence and/orquantity of a specific breast cancer marker in the sample). In anotherexample, sample verification is achieved through a combinatorial (i.e.multiple marker) immunoassay targeting various cytokeratins, which canbe detected as a panel of cytokeratins specifically expressed in mammarytissue sample. (See, Nagle, J., Histochem. Cytochem. 34:869-881, 1986,incorporated herein by reference in its entirety). One or more of thesecytokeratins (e.g. K5, K8, K18 and K19) can be simultaneously orindependently measured in the context of a breast cancer assay, and thelevel of expression of the subject cytokeratin(s) can yield informationconcerning the presence or status of breast cancer in a patient. (Seefor example, Focus, Harvard University News Office, Mar. 21, 1991, pp.2-3; and Lee, Proc. Natl. Acad. Sci. USA 88:1991, each incorporatedherein by reference in its entirety).

EXAMPLE 5 Immunoassay for Human Mammary Epithelial Antigens inBiological Samples From Mammary Fluid

Human mammary epithelial antigens (HME-Ags) are glycoprotein componentsof the human milkfat globule (HMFG) and of the membrane of the breastepithelial cell, which are released by breast tumors and not by normalbreast tissue. (Ceriani et al., Proc. Natl. Acad. Sci. USA 74:582-586,1977, incorporated herein by reference in its entirety). In the presentexample, several HME-Ags, having molecular weights of 150, 70, and 45kilodaltons, are detected and measured using specific anti-HMFG oranti-human mammary epithelial (α-HME) probes prepared and employed asdescribed by Ceriani et al., Proc. Natl. Acad. Sci. USA 79:5420-5425,1982 (incorporated herein by reference in its entirety).

To begin the assay, biological samples from mammary fluid collected onnitrocellulose filters coupled with a breast pump 10, 10′ as generallydescribed above are eluted electrophoretically into phosphate bufferedsaline to provide a test sample, according to standard methods.Alternatively, whole mammary fluid or other types of biological samplesobtained from mammary fluid can be constituted in an appropriate mediumor mixture to provide a test sample for the assay. Once the test sampleis thus provided, it is then assayed according to the HME-Agsradioimmunoassay (RIA) methods described in Ceriani et al., BreastCancer Res. Treat. 15:161-174, 1990 (incorporated herein by reference inits entirety).

Briefly, the RIA includes two preliminary treatments of the biologicalsamples to separate interfering factors: a centrifugation step toseparate out any fat present, and a second, precipitation step toprecipitate potential immunocomplexes using polyethyleneglycol (PEG).The next steps comprise the assay proper, where HMFG antigen bound to asolid support (microtiter plates) is presented to stoichiometric orlesser amount of the α-HME antibody probe, and binding of the α-HME iscompeted by the biological samples from mammary fluid preliminarilytreated as above. The amount of α-HME bound to HMFG antigen on the solidphase is determined in a final step by detection of the α-HME antibodyprobe by radioiodinated, affinity-purified rabbit anti-mouseimmunoglobulin.

Solutions used in the assay are as follows: i) Phosphate buffered saline(PBS): 176 ml 0.05M KH₂PO₄, 608 ml 0.05M Na₂HPO₄, and 8 g NaCl broughtup to 1000 ml in H₂O (pH7.4). ii) RIA buffer: 0.1% BSA, in 0.3%Triton-X-100 (Research Prod. International Corp., Mount Prospect, Ill.)plus 0.05% sodium azide in PBS. iii) Detergent buffer: 0.3% Triton-X-100plus 0.05% sodium azide in PBS. iv) Buffered polyethylene glycol (PEG):6.6% PEG (M.W. 8000) (Sigma) plus 0.05% sodium azide in PBS)¹²⁵I-labeled affinity-purified rabbit anti-mouse immunoglobulin(Rα-mouse Ig) (Antibodies, Inc., Davis, Calif.), radioiodinated by thechloramine-T procedure as reported (Ceriani et al., Proc. Natl. Acad.Sci. USA 79:5420-5425, 1982) and diluted to 4×10⁶ cpm/ml, in RIA buffer.Rabbit polyclonal anti-HMFG antibodies or rabbit anti-human mammaryepithelial antibodies (α-HME) were prepared and assayed as described(Id.).

To prepare a standard curve for evaluating assay results, controlsamples from normal human mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed,eluted test sample, or alternatively provided as normal whole mammaryfluid or other selected type of sample obtained from normal mammaryfluid, constituted in an appropriate medium or mixture to provide asuitable control assay sample) are centrifuged for 7 min at 10,240 rpmat 10° C. The upper white band formed at the top of the sample (if thereis one) is discarded. Fresh 100 μg protein/ml solution of lyophilizeddilipidated HMFG (Ceriani et al., Proc. Natl. Acad. Sci. USA 74:582-586,1977) in detergent buffer is prepared and sonicated at 10 secondintervals for a total of 4 minutes (10 sec. of sonication, followed by a10 sec. silent period) using a double step micro tip horn at 25 watts ona Sonifier Cell Disrupter 185 (Branson, Danbury, Conn.) at 4° C. HMFGsolutions at concentrations of 0, 10, 33.3, 100, 333.3, and 1000 ngprotein/ml are prepared in spun female sera, and 3 aliquots of 180 μl ofeach HMFG level in normal female sera are pipetted into 400 pipolyethylene microcentrifuge tubes (West Coast Sci. Emeryville, Calif.).150 μl of 6.6% PEG solution is added to each microcentrifuge tube, andthe tubes are incubated overnight on a rotating shaker at roomtemperature.

Test samples are processed in a comparable manner, by centrifuging300-350 μl of the eluted nitrocellulose filtrate in solution (or,alternatively, of mammary fluid or other assay sample alternative) in a400 μl microcentrifuge tube for 5-7 min. at 10,240 rpm at 10° C. Themicrocentrifuge tubes are then cut with a razor blade below the whiteband formed by the sera (if there was one) and 180 μl of remaining serais transferred to a new microcentrifuge tube. 150 μl of a 6.6% PEGsolution is then added to each microcentrifuge tube, and the tubes areincubated overnight on a rotating shaker at room temperature.

Day two

(1) α-HME is diluted to its appropriate concentration in detergentbuffer. The antibody solution has stoichiometric or lesser amounts ofA-HME to 6 ng HMFG protein equivalent (prot. eq.). Six ng of HMFG iscovalently bound to microtiter plates by the methylated BSA procedurepreviously described by Ceriani, in Monoclonal Antibodies and FunctionalCell Lines, pp.398-402, Kennet et al (eds), Plenum Press, New York,1984, incorporated herein by reference in its entirety.

(2) To process test samples and control samples on the second day,microcentrifuge tubes are centrifuged for 7 min. at 10,240 rpm at 10° C.in a SHMT rotor with a Sorvall RC5C centrifuge. In triplicate, 55 μl ofsupernatant is pipetted into empty microtiter plate wells (Dynatech,Alexandria, Va.), and any precipitate pelleted is left undisturbed. 25μl of 6.6% PEG solution is added to each well. 30 μl of α-HME diluted indetergent buffer is also added to each well, and a non-porous Scotch®tape is placed over the wells to avoid evaporation. The microtiter plateis then incubated overnight at room temperature on a rotary shaker.

Day Three

The microtiter plates are centrifuged (3000 r.p.m.) for 30 minutes atroom temperature to decant suspended perceptible matter. 50 μl of RIAbuffer is added to wells of microtiter plates containing 6 ng HMFG andaspirated off after 5 minutes.

The total contents of microtiter plates from 1), save for anyprecipitation induced by the PEG and already pelleted, are carefullytransferred to the wells of another set of microtiter plates containing6 ng HMFG per well (Day 2,1), above.

The microtiter plates are incubated for 3 hours with rotating agitationat room temperature. The plates are washed 5 times with RIA buffer usingDynadrop SR4 automatic dispenser form Dynatech. 50 μl of theradioiodinated affinity-purified rabbit anti-mouse immunoglobulindiluted in RIA buffer is then adder per well. The plate is covered withtape and incubated with rotating agitation for 2 hours at roomtemperature. The plate is washed 5 times with RIA buffer. Wells are cutand counted in a gamma counter.

The results of these assays will yield important information concerningthe presence and/or status of cancer in patients, comparable in scopeand value to the data provided by serum assays conducted for the HME-Agbreast cancer marker by Ceriani et al, Breast Cancer Res. Treat.15:161-174, 1990. By selecting patient and control samples anddeveloping and evaluating comparative data according to the proceduresfollowed by Ceriani and his coworkers, the assay methods of theinvention will also be readly adapted for use in direct clinicalapplications to determine both prognostic and treatment relatedvariables in breast cancer patients. Reagents and conditions for theassays can of course be substituted or adjusted depending on a varietyof anticipated variables, by applying well known immunological methodsand principles.

EXAMPLE 6 Competitive Radioimmunoassay for Non-Penetrating Glycoproteinin Biological Samples From Mammary Fluid

This competitive radioimmunoassay is based on the displacement bybreastepithelial antigens contained in biological samples from mammary fluidobtained according to the methods of the invention of the binding ofstoichiometric or lesser quantities of the monoclonal antibody Mc5 to asolid-phase-bound antigen known as non-penetrating glycoprotein (NPGP)contained in HMFG. HMFG is bound to a solid support and exposed to theMc5 antibody during an incubation period allowing the antibody to bindthe NPGP antigen in solid phase-bound HMFG. The presence and/or level ofNPGP in the biological sample is ultimately examined by ability of thesample to compete for Mc5 binding to the NPGP antigen in the solidphase-bound HMFG, as detected and/or measured using a radiolabeled goatanti-mouse antibody to bind and label the Mc5 antibody probe.

Buffer and other solutions and reagents in this example are generallythe same as those used for the HME-Ags polyclonal antibodyradioimmunoassay described in Example 4, above. To provide test samplesfor the assay, biological samples from mammary fluid contained onnitrocellulose filters are eluted electrophoretically into phosphatebuffered saline, according to standard methods. Alternatively, wholemammary fluid or other types of biological samples obtained from mammaryfluid can be constituted in an appropriate medium or mixture to providea test sample for the assay. Once the test sample is thus provided, itis then assayed according to the NPGP/Mc5 radioimmunoassay (RIA) methodsdescribed in Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990(incorporated herein by reference in its entirety), as follows:

400 μl of pooled normal female mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed,eluted test sample, or alternatively provided as normal whole mammaryfluid or other types of biological samples obtained from normal mammaryfluid constituted in an appropriate medium or mixture to provide a testsample) to provide a suitable control sample, which is diluted to 2.4 mlusing RIA buffer at a 1:6 concentration.

A 500 μg/ml solution of lypholized HMFG is prepared in 1× PBS with 0.3%Triton-X-100, 0.05% sodium azide, and sonicated using a double stepmicro tip horn at 25 watts on a Sonifier Cell Disrupter 185 (Branson,Danbury, Conn.) for 4 minutes (10 sec. sonication, 10 sec. silentperiod, at 4° C.).

Solutions to prepare a standard curve are prepared using the 2.4 ml 1:6normal female serum and increasing amounts of HMFG (0, 0.25, 2.5, 25, 50μg/ml HMFF, as described above in Example 4).

Each test assay sample is diluted 1:6 with RIA buffer (40 μL of serum to200 μL RIA buffer) to form a diluted test assay sample, and vortexed.

Mc5 stock solution is prepared so that it contains less thanstoichiometric amounts of antibody to 100 ng protein/well of HMFGcovalently bound to microtiter plates prepared as previously describedby Ceriani, in Monoclonal Antibodies and Functional Cell Lines, pp.398-402, Kennet et al. (eds), Plenum Press, New York, 1984, incorporatedherein by reference in its entirety

200 μl RIA buffer are added to each well of 100 ng HMFG and thenaspirated after 5 minutes.

To prepare a standard curve, 30 μl of HMFG standardizing solutions (asin 3 above) are added in quadruplicate to a 100 ng protein/well HMFGmicrotiter well. 30 μl of diluted test assay sample (or, alternatively,of mammary fluid or other assay sample alternative) are added intriplicate to 100 ng/well HMFG microtiter wells.

To each well 20 μl of the Mc5 stock solution is added.

Microtiter plates are covered with nonporous Scotch® tape and incubatedovernight at room temperature on a rotating agitator.

The next day the wells are aspirated and washed 5 times with RIA buffer.

To each well 50 μL of 200,000 cpm/50 μl ¹²⁵i-goat anti-mouse antibodyare dispensed. The wells are covered with nonporous tape and placed on arotating agitator for 3 hours at room temperature.

Wells are washed 5 times with RIA buffer.

Each well is cut and the radioactivity is counted using a gamma counter.

The results of these assays will yield important information concerningthe presence and/or status of cancer in patients, comparable in scopeand value to the data provided by serum assays conducted for the NPGPbreast cancer marker by Cerianiet al., Breast Cancer Res. Treat.15:161-174, 1990. By selecting patient and control samples anddeveloping and evaluating data according to the well known proceduresfollowed by Ceriani and his coworkers, the assay methods of theinvention will be readily adapted for use in direct clinicalapplications to determine both prognostic and treatment relatedvariables in breast cancer patients. As will be understood by thoseskilled in the art, reagents and conditions for the assays can besubstituted or adjusted depending on a variety of anticipated variables,according to well known immunological methods and principles.

EXAMPLE 7 Solid Phase Immunoassay for Mucinous Carcinoma AssociatedAntigen in Mammary Fluid

This example uses a sensitive, solid phase immunoassay to detect themucinous carcinoma associated antigen (MCA) in biological samples frommammary fluid obtained according to the methods of the invention. MCAconcentrations are determined using an antibody-bead immunoassay kitprovided by Hoffinan-La Roche (Basel, Switzerland), and using thereagents and procedures provided by the manufacturer and described infurther detail in Eskelinen et al., Anticancer Res. 9:437-440, 1989.Briefly, test assay samples of whole mammary fluid and standards arefirst incubated with MCA monoclonal antibody beads and then, afterappropriate washings, enzyme (horseradish peroxidase) labeled secondaryantibody is added. During the second incubation the anti-MCA enzymeconjugates are attached to the antibody antigen complex on the beads.Excess conjugates are removed by washing and, finally, enzyme substrateare added and the color formed is recorded.

The solid phase assay format presented in this example can be adaptedfor use in a wide array of other assays to detect and/or measure othercancer markers besides the MCA marker, with enhanced sensitivity. Inaddition, the results of these assays can be evaluated along with thoseof complementary assays detecting and/or measuring different markers toyield more precise information concerning the presence and/or status ofcancer in patients, as exemplified by the combinatorial MCA/CA 15-3assays described by Eskelinenet al., Anticancer Res. 9:437-440, 1989;see also Eskelinen et al., Anticancer Res. 8:665-668, 1988, eachincorporated herein by reference in its entirety.

EXAMPLE 8 Western Analysis of Proteins From Cellular Fractions of HumanMammary Fluid Using Polyclonal and Monoclonal Antibody Probes to DetectVasopressin

A variety of assays are provided by the present invention that focus oncellular samples from human mammary fluid. In general, these assays relyon isolation by standard separation methods (e.g. centrifugation,sucrose gradient, etc.) of cells, membranes or other cell componentsfrom whole mammary fluid expressed according to the above methods.Biological samples containing whole cells from expressed mammary fluidare particularly useful for cytological and cytochemical examination todetect and evaluate breast cancer in patients. Biological samplescontaining purified cell membrane fractions from human mammary fluid areparticularly useful in this context, for example to detect and/ormeasure breast cancer markers that are expressed by alveolar-ductalcells as intracellular or membrane bound proteins and are therefore notas readily detected in liquid fractions of mammary fluid as secretedproteins.

The present example focuses on assays for detecting the peptide hormonevasopressin in biological samples from mammary fluid, using methodsadapted from North et al, Breast Cancer Res. Treat. 34:229-235, 1995.Specifically, this assay uses a test sample of crude protein isolatedfrom a pooled sample of cells obtained from expressed mammary fluid. Thecells are separated from whole mammary fluid according to standardmethods, and crude protein is extracted from the cells by sonication in100 volumes of 0.1 M HCl. The resuling protein suspensions are thencentrifuged at 1500× g for 10 min. at ambient temperature, and solubleprotein is precipitated with 40% TCA. This protein is pelleted bycentrifugation at 10,000× g for 2 min. TCA is then removed from pelletsby washing (×2) with ether. Protein is resuspended in 0.1 M Tris HCl(pH8.7), reduced with mercaptoethanol at 100° C. for 5 min. (and in somecases S-alkylated with N-ethyl maleimide), and subjected to SDS-PAGEelectrophoresis on 15% gels at pH 9.3 using the method of Laemeli,Nature 227:680-685, 1970, incorporated herein by reference in itsentirety. Separated proteins are then electrophoretically transferredwith 20 mM Tris glycine (pH 8.0) to Immobilon PVDF membranes (Millipore,Bedford, Mass.). These membranes are blocked with a 5% non-fat milksolution, washed (1×15 min., 2×5 min.) with PBS containing 0.5% Triton,and incubated with preparations of mouse monoclonal antibody to VP-HNP,with rabbit polyclonal antibodies to VP, with rabbit polyclonalantibodies to VAG, or with ubiquitous mouse or rabbit IgG (negativecontrols) (for description of antibodies and antibody preparation seeNorth et al., Breast Cancer Res. Treat. 34:229-235, 1995, incorporatedherein by reference in its entirety), for 1 h at ambient temperature.Following a second wash in PBS-Triton (1×15 min., 2×5 min.), themembranes are treated, respectively, with goat anti-mouseIgG-horseradish peroxidase conjugate or goat anti-rabbit IgG-horseradishperoxidase conjugate for 1 h, and then washed with PBS-Triton (1×15min., 4×5 min.). Immunoreactive proteins are visualized using an ECLWestern Blotting Detection System with exposure of x-ray film from 10seconds to 5 min. Prestained SDS-PAGE standard proteins are employed asmolecular size markers.

Recent studies suggest that vasopressin is universally expressed inbreast carcinoma and is absent from normal breast cells. North et al.,Breast Cancer Res. Treat. 34:229-235, 1995. These and other resultsindicate that vasopressin and its relatives are important breast cancermarkers that can be readily detected using immunological assays ofproteins isolated from breast tumor cells. Accordingly, the results ofthe present example using cell samples isolated from human mammary fluidare also expected to yield important information concerning the presenceand/or status of cancer in patients.

EXAMPLE 9 Quantification of Carcinoembryonic Antigen in BiologicalSamples From Mammary Fluid by Dot Immunoblotting Assay

Among the more sensitive assays of the invention, useful for measuringlow levels of breast cancer markers and for detecting markers when onlysmall volumes of expressed mammary fluid are available, is the dotimmunoblotting assay. In the present example, carcinoembryonic antigen(CEA) is measured in whole mammary fluid using an Elmotech anti-CEAmonoclonal antibody kit (Mochid Pharmaceutical Co., Tokyo, Japan) in adot blot assay format. Briefly, anti-CEA monoclonal antibody is dilutedto appropriate concentrations and coated on the plastic film. Aliquots(5 μl) of either standard CEA solution (0, 100, 200, and 500 ng/ml), orof the whole mammary fluid assay sample, are smeared on the immobilizedfilm. Assay standards are prepared from purified antigen preparations,in accordance with the Elmotec kit manufacturer's instructions. Ifnecessary, 1000 ng/ml CEA solution is also used as a standard. Afterdrying at room temperature, the film is exposed to peroxidase-conjugatedanti-CEA antibody for 20 min at room temperature. The film is thenwashed extensively with 1 M saline containing 0.5% (v/v) Tween 20. Theenzyme reaction is visualized using tetramethylbenzidine as a chromogen.The developing solution consists of 0.05 mM tetramethylbenzidine and0.01% hydrogen peroxide in Mcllvain buffer (0.1% M phosphate-citratebuffer), pH 5.0, containing 10% methanol. The concentration of CEA inthe mammary fluid assay sample is determined by comparing the colorintensities with a corresponding standard.

The assay disclosed in the present exanple, and related assaysincorporating antibodies to other tumor markers besides CEA, areparticularly useful for measuring low levels of breast cancer markersand for detecting markers in limited sample volumes. The results ofthese assays will yield important information to determine bothprognostic and treatment related variables in breast cancer patients. Aswill be understood by those skilled in the art, reagents and conditionsfor the assays can be substituted or adjusted depending on a variety ofanticipated variables, according to well known immunological methods andprinciples.

EXAMPLE 10 Detection of Procathepsin D and Cathepsin D Activity inBiological Samples From Mammary Fluid

Cathepsin D is a lysosomal aspartic proteinase that has been studiedintensively as a marker for cancer processes necessary for metastasis.In the present example, polyclonal antibodies against procathepsin D areused to immunoprecipitate and immunochemically detect proteins fromwhole mammary fluid or cell lysates from mammary fluid, generallyaccording to the methods disclosed in Vetvicka et al., Biochem. Mol.Biol. Int'l. 30:921-928, 1993 (incorporated herein by reference in itsentirety). Alternatively, or as a complementary assay, the proteaseactivity of cathepsin D is detected, also according to the methodsdisclosed in Vetvicka et al. (Id.). Briefly, pooled whole mammary fluid(preferably 3 ml if available) is diluted with 3 ml of buffer A (50 mMTris.HCl, 5 mM CaCl₂, 1 mM MgCl₂, 500 mM NaCl pH 7.2). The suspension iscentrifuged for 30 minutes at 10,000 g. The resulting water phase iscentrifuged again under the same conditions. The soluble part (total ofapproximately 4.5 ml) is loaded on a 1 ml column of Concanavalin ASepharose (Pharmacia, Uppsala, Sweden) equilibrated in buffer A, andafter washing with buffer A the retained proteins are eluted using 0.75M methyl a-D-mannopyranoside. The fractions (250 μl) are analyzed forcathepsin D activity using the ¹⁴C hemoglobin assay as described by Linet al., J. Biol. Chem. 264:4482-4489, 1989 (incorporated herein byreference in its entirety), by western blots and by silver-stainedelectrophoresis. The inhibition of human milk procathepsin D isaccomplished by adding 2 μl of 1 mM pepstatin A (Boehringer Manhein,Germany) dissolved in methanol to the reaction mixture.

This assay provides but one example of many possible embodiments of theinvention that incorporate known biochemical assays, in addition to, orsupplemental to immunological assays, to evaluate biological samplesfrom mammary fluid to determine cancer related variables. Thefundamental methods provided herein for obtaining samples from humanmammary fluid render these assays readily adaptable for widespreadclinical application to detect and/or measure the activity of a subjectbreast cancer marker within a non-invasive screening protocol.

Those with ordinary skill in the art will appreciate that otherembodiments and variations of the invention are possible which employthe same inventive concept described above. Most particularly, a wideand rapidly expanding array of useful breast cancer markers (includingproteins, DNA and RNA sequences and other markers) and probes (includingimmunological, nucleotide and biochemical probes) are readily availablefor adaptation and use within the methods and kits of the invention.These markers and probes are described or referenced to a large extentin the literature cited and incorporated within the present disclosure,or are elsewhere published in the literature or well known in the art.Among these known and emerging markers and probes, useful exampleswithin the invention include Her 2 (also known as erbB-2 and neu). Her 2is a transmembrane glycoprotein growth factor receptor of the EGFreceptor family encoded by a gene located on chromosome 17q, a region offrequent amplification in breast cancer cell lines. This marker ishighly predictive of breast cancer and can be detected in cellularsamples of the invention using known nucleotide probes to detect geneticdefects in Her 2, or to detect and/or measure mRNA to determineoverexpression of Her 2 linked to increased proliferation of cancercells. (See for example, Visscher et al., In Weinstein and Graham (eds)Advances in Pathology and Laboratory Medicine, vol. 5, St. Louis, MosbyYuear Book, 1992, pp. 123-161; Barbareschiet al., Am. J. Clin. Pathol.98:408-418, 1992; Slamonetal., Science 235:177-182, 1987; eachincorporated herein by reference in its entirety). Protein levels of Her2 are also readily detected using available immunological probes. (Forreview see Porter-Jordan et al, Hematol. Oncol. Clin. North Amer.8:73-100, 1994 and articles cited on page 80 therein, each incorporatedherein by reference in its entirety). Additional markers for use withinthe invention include EGF and the EGF receptor, for which immunologicaland non-immunological probes and assay methods readily adaptable withinthe invention are characterized in detail at page 80-81 of Porter-Jordanet al., Hematol. Oncol. Clin. North Amer. 8:73-100, 1994 and in thereferences cited therein, each incorporated herein by reference in itsentirety. Additional examples of proliferation markers, growth factorsand receptors, proteases, adhesion factors, angiogenic factors,oncogenes and tumor suppressor genes that provide useful breast diseasemarkers and probes within the methods and kits of the invention includeKi67 Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen,Transforming Growth Factor, Tissue Plasminogen Activator, Insulin GrowthFactor Receptors, Collagenase Type IV, Laminin Receptor, Integrins, p53,rb, nm23, ras, c-myc, myb, Heat Shock Proteins, Prolactin,Neuron-Specific Enolase, IR-14, KA 1, KA 14, AlphaLactalbumin, Actin,IL-10, S-100 protein, Vimentin, Epithelial Membrane Antigen, bc1-2,CA15-3, CA 19-9, Tn Antigen, Alpha-lactalbumin, LASA, Gal-GalNAC,GCDFP-15, Le(y)-Related Carbohydrate Antigen, CA 125, uPA, uPA relatedantigens and complexes, uPA Receptor, PA1-1 and PA1-2,Beta-glucuronidase, CD31, CD44 splice variants, blood group antigensincluding ABH, Lewis, and MN, and genetic lesions or altered expressionlevels of CCND1, EMS1, BRCA1 and BRCA2 genes, and many others, for whichimmunological and non-immunological binding partners, probes and assaymethods are known and readily adaptable within the invention.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and may be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

What is claimed is:
 1. A mammary fluid collection reservoir forcollection, handling and processing biological samples of mammary fluidcomprising: a rigid tube or vial adapted for removable, sealableengagement in a mammary fluid collection device to establish a gaseousand fluid connection between a breast engaging member of the collectiondevice and an inner lumen of the rigid tube or vial, said rigid tube orvial having a top end defining a primary opening for collecting andaccessing the mammary fluid sample and a cylindrical outer wall defininga reservoir lumen closed at a bottom end thereof, said outer walldefining one or more air ports that communicate between the outer walland the lumen of the rigid tube or vial, further comprising closuremeans for sealably closing the tube or vial after the mammary fluid isintroduced therein and the tube or vial is disengaged from the mammaryfluid collection device, to seal the tube or vial and prevent samplecontamination and spillage during handling and processing of thebiological sample.
 2. The mammary fluid collection reservoir of claim 1,wherein the reservoir comprises a removable fluid reservoir member of asample collection housing of a mammary fluid collection device.
 3. Themammary fluid collection reservoir of claim 2, wherein the rigid samplecollection tube or vial removably connected with an outer casing memberof the sample collection housing of the mammary fluid collection device.4. The mammary fluid collection reservoir of claim 3, wherein thereservoir is adapted for removable, sealable connection with said outercasing member of the housing, to form an airtight coupling therewith. 5.The mammary fluid collection reservoir of claim 4, wherein the reservoiris a cytology vial sealably connectable with the outer casing member toform the airtight coupling.
 6. The mammary fluid collection reservoir ofclaim 2, wherein the reservoir is adapted to be removably nested withinan outer casing member of the sample collection housing to form asubstantially airtight contact between an inner wall of the casingmember and an outer wall or top or bottom end of the reservoir member.7. The mammary fluid collection reservoir of claim 2, wherein the outerwall of the reservoir is provided with a circumferential ridge, fin orO-ring.
 8. The mammary fluid collection reservoir of claim 1, whereinsaid closure means comprises a cap adapted to sealably engage a top endof the fluid-retaining reservoir.
 9. The mammary fluid collectionreservoir of claim 8, wherein a top end of the tube or vial is providedwith complementary threads to receive a conventional cap that iscomplementarily threaded to sealably engage the top end of the tube orvial.
 10. The mammary fluid collection reservoir of claim 8, whereinsaid closure means includes secondary closure means for closing said oneor more air ports.
 11. The mammary fluid collection reservoir of claim10, wherein said secondary closure means includes a secondary closuredevice selected from a plug, cap or adhesive cover sized and constructedto engage or adhere to the outer wall of the reservoir at or surroundingthe one or more air port openings to form a secondary closure.
 12. Themammary fluid collection reservoir of claim 10, wherein said secondaryclosure means comprise a combined closure and labeling device whichfunctions as a secondary closure mechanism to seal the one or more airports of the reservoir and as a labeling template to provide a writingsurface for sample labeling.
 13. The mammary fluid collection reservoirof claim 12, wherein said combined closure and labeling device is alabeling tab or sticker for application to the outer wall of thereservoir to seal the one or more air ports after the sample iscollected.
 14. The mammary fluid collection reservoir of claim 13,wherein said tab or sticker has a first, closure-forming surface forapplication over the one or more air ports to form a seal byjuxtaposition or adhesive contact with the outer wall of the reservoir,and a second, labeling surface opposite the closure-forming surface madeof a blank template material for imprinting written information thereon.15. The mammary fluid collection reservoir of claim 12, wherein saidsecondary closure means is pre-attached to the reservoir member in afirst, open configuration and is adapted to be manually repositioned orotherwise manipulated after sample collection to a second, closedconfiguration to form a seal or closure against the one or more airports and provide an and exposed writing surface for sample labeling.16. A method for handling or processing a biological sample of mammaryfluid or a component thereof for use in a diagnostic assay to detect orquantify a breast disease marker in said sample comprising the step of:providing or obtaining said biological sample of mammary fluid orcomponent thereof in a fluid-retaining reservoir having a top enddefining a primary opening for access to the sample and an outerreservoir wall defining one or more air ports that communicate betweenthe outer wall and an inner lumen of the reservoir, said reservoircomprising a rigid tube or vial adapted for removable, sealableengagement in a mammary fluid collection device to establish a gaseousand fluid connection between a breast engaging member of the collectiondevice and an inner lumen of the rigid tube or vial, said reservoirfurther comprising closure means for sealably closing the tube or vialafter the mammary fluid is introduced therein and the tube or vial isdisengaged from the mammary fluid collection device, to seal the tube orvial and prevent sample contamination and spillage during handling andprocessing of the biological sample.
 17. The method according to claim16, wherein the closure means comprises a cap adapted to sealably engagea top end of the fluid-retaining reservoir.
 18. The method according toclaim 17, wherein a top end of the tube or vial is provided withcomplementary threads to receive a conventional cap that iscomplementarily threaded to sealably engage the top end of the tube orvial.
 19. The method according to claim 17, wherein the closure meansfurther comprises secondary closure means for sealably closing the oneor more air ports after said sample is introduced into said reservoir.20. The method according to claim 19, wherein said secondary closuremeans comprise an adhesive seal or sticker sized and constructed toadhere to an outer wall of the reservoir surrounding an air portopening.
 21. The method according to claim 19, wherein said secondaryclosure means comprises a combined closure and labeling device whichfunctions as a secondary closure mechanism to seal the one or more airports of the fluid-retaining reservoir and as a labeling template toprovide a writing surface for sample labeling.
 22. The method accordingto claim 19, wherein said secondary closure means comprises a combinedclosure and labeling tab or sticker which is adapted to be directlyapplied to seal the one or more air ports after sample collection havinga first, closure-forming surface for application over the air port toform a seal by juxtaposition or adhesive contact with an outer wall ofthe fluid-retaining reservoir, and a second, labeling surface oppositethe closure-forming surface made of a blank template material suitablefor receiving a stable, ink or graphite imprint thereon.
 23. The methodaccording to claim 22, wherein said first, closure-forming surface bearsan adhesive coating resistant to disruption by contact with aqueoussolutions.
 24. The method according to claim 19, wherein said secondaryclosure means comprises a combined closure and labeling tab or stickerwhich is pre-attached to the fluid-retaining reservoir in a first, openconfiguration and is adapted to be manually repositioned or otherwisemanipulated after sample collection to a second, closed configuration toform a seal or closure against the one or more air ports.
 25. The methodaccording to claim 24, wherein said secondary closure means comprises anadhesive tab or strip folded in the open configuration to form an innerlayer affixed to the reservoir proximate to the one or more air portsand an outer layer folded over the inner layer, said outer layerproviding a first, closure-forming surface and a second, labelingsurface, wherein the outer layer can be unfolded away from the innerlayer and wrapped around the reservoir so that the closure-formingsurface covers the one or more air ports to form a fluid-resistantclosure and the labeling surface faces outward for recordation of sampledata.
 26. The method according to claim 25, wherein the outer layer isoptionally secured in a folded-back position against the inner layer byadhesive engagement of the labeling surface with the inner layer. 27.The method according to claim 26, wherein said first, closure-formingsurface bears an adhesive coating that is protected in the openconfiguration by folding of an end segment of the outer layer bearingthe adhesive coating back, so that the closure forming surface providesa protective surface to shield the adhesive prior to closure, wherebythe end segment is adapted to be lifted and pulled outward to unfold theend segment to separate the adhesive coating on the closure-formingsurface from the protective surface and to release the outer layer fromthe inner layer for closing of the one or more air ports.
 28. The methodaccording to claim 1, wherein the fluid-retaining reservoir is amodified cytology vial.
 29. The method according to claim 1, wherein thefluid-retaining reservoir comprises a removable fluid reservoir memberof a sample collection housing of a mammary fluid collection device. 30.The method according to claim 29, wherein the rigid tube or vial isremovably connected with an outer casing member of the sample collectionhousing of said collection device.
 31. The method according to claim 30,wherein the fluid-retaining reservoir is adapted for removable, sealableconnection with said outer casing member of said housing to form anairtight coupling therewith.
 32. The method according to claim 31,wherein the fluid-retaining reservoir is a cytology vial sealablyconnectable with said outer casing member to form an airtight couplingtherewith.
 33. The method according to claim 32, wherein thefluid-retaining reservoir is adapted to be removably nested within saidcasing member to form a substantially airtight contact between an innerwall of the casing member and an outer wall, or a top or bottom end, ofthe reservoir member.
 34. The method according to claim 1, wherein anouter wall of the fluid-retaining reservoir bears a circumferentialridge, fin or O-ring.
 35. The method according to claim 34, wherein thefluid-retaining reservoir comprises a removable fluid reservoir memberof a sample collection housing of a mammary fluid collection device andthe circumferential ridge, fin or O-ring is adapted to engage and make acircumferential airtight seal against an inner wall of a casing memberof a sample collection housing of said device.
 36. The method accordingto claim 1, wherein said mammary fluid sample or component thereofincludes whole cells or cell fragments.
 37. The method according toclaim 1, further comprising the step of accessing said sample withinsaid reservoir to transfer or process the sample for detection orquantification of said breast disease marker.
 38. The method accordingto claim 31, further comprising the step of transferring said sample toa second reservoir or other sample container or template for processingthe sample to detect or quantify said breast disease marker.
 39. Themethod according to claim 38, wherein said step of processing saidsample to detect or quantify said breast disease marker comprisesstaining cells or cell fragments from said sample with a cytologicalstain to detect a cytological marker.
 40. The method according to claim31, further comprising the step of processing said sample to detect orquantify said breast disease marker by microscopic examination ofstained cells or cell fragments from said sample.
 41. The methodaccording to claim 1, wherein said biological sample includes one ormore components selected from the group consisting of whole mammaryfluid, whole cells, cell fragments, cell membranes, purified proteins,bulk proteins, glycoproteins, peptides and polynucleotide components ofa primary mammary fluid sample.
 42. The method according to claim1,wherein the breast disease marker is a breast cancer marker.
 43. Themethod according to claim 1, wherein the breast disease marker isselected from the group consisting of a protein, a peptide, aglycoprotein, a lipid, a DNA polynucleotide and an RNA polynucleotide.44. The method according to claim 1, wherein the breast disease markeris selected from the group consisting of Ki67 Growth Factor, Cyclin D1,Proliferating Cell Nuclear Antigen, Transforming Growth Factor TissuePlasminogen Activator, Insulin Growth Factor Receptors, Collagenase TypeIV, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat ShockProteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14,Alpha-Lactalbumin and Actin.
 45. The method according to claim 1,whereinthe breast disease marker is selected from the group consisting of CEA,HMFG, MCA, vasopressin and cathepsin.
 46. The method according to claim1, further comprising the step of detecting or quantifying said breastdisease marker by ELISA immunoassay, immunoprecipitation assay, or solidphase immunoassay.
 47. The method according to claim 1, wherein saidbiological sample comprises an oxytocin-induced sample of mammary fluidor a component thereof.
 48. The method according to claim 47, whereinsaid biological sample comprises whole cells or cell fragments from saidoxytocin-induced sample of mammary fluid.